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Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex

  • John Simms
    John Simms
    Aston University, Birmingham B4 7ET, U.K.
    Coventry University, Priory Street, Coventry CV1 5FB, U.K.
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  • , 
  • Romez Uddin
    Romez Uddin
    Aston University, Birmingham B4 7ET, U.K.
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  • , 
  • Thomas P. Sakmar
    Thomas P. Sakmar
    The Rockefeller University, 1230 York Avenue, New York, New York 10065, United States
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  • , 
  • Joseph J. Gingell
    Joseph J. Gingell
    University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
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  • , 
  • Michael L. Garelja
    Michael L. Garelja
    University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
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  • , 
  • Debbie L. Hay
    Debbie L. Hay
    University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
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    Orcidhttp://orcid.org/0000-0002-9558-5122
  • , 
  • Margaret A. Brimble
    Margaret A. Brimble
    University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
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    Orcidhttp://orcid.org/0000-0002-7086-4096
  • , 
  • Paul W. Harris
    Paul W. Harris
    University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
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  • , 
  • Christopher A. Reynolds
    Christopher A. Reynolds
    University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
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  • David R. Poyner*
    David R. Poyner
    Aston University, Birmingham B4 7ET, U.K.
    *E-mail: [email protected]. Telephone: +44 (0)121 204 3997.
    More by David R. Poyner
    Orcidhttp://orcid.org/0000-0003-1590-112X
Cite this: Biochemistry 2018, 57, 32, 4915–4922
Publication Date (Web):July 13, 2018
https://doi.org/10.1021/acs.biochem.8b00502

Copyright © 2018 American Chemical Society. This publication is licensed under CC-BY.

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Abstract

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Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

Calcitonin gene-related peptide (CGRP) is an abundant sensory neuropeptide; it is known to be a very potent vasodilator and is implicated in migraines. It is part of the calcitonin superfamily of peptides that also includes adrenomedullin, adrenomedullin 2/intermedin, and amylin. (1,2) The peptides all bind to family B/secretin-like G protein-coupled receptors, either the calcitonin receptor or the calcitonin receptor-like receptor (CLR). However, for high-affinity binding for all of these ligands apart from calcitonin, it is necessary for the receptor to form a complex with a second transmembrane protein from the receptor activity-modifying protein (RAMP) family with only three members. The CGRP receptor is formed from CLR and RAMP1, although CGRP will also bind with high affinity to the complex of the calcitonin receptor and RAMP1. (1)
The binding of endogenous peptide ligands to family B GPCRs generally follows the two-domain model in which the N-terminal tail of the peptide interacts with the transmembrane (TM) domain of the receptor whereas the C-terminal tail binds to the extracellular domain (ECD) of the receptor. (3) Recently, a number of structures of full length family B GPCRs have been published, namely, for the glucagon and glucagon-like peptide 1 (GLP-1) receptors. (4−8) The full length calcitonin receptor has been visualized by cryo-electron microscopy, but the ECD of the receptor was very flexible and so could not be resolved. The bound salmon calcitonin peptide was also poorly resolved, and its binding mode was refined by only rigid body fitting of the calcitonin receptor ECD structure [Protein Data Bank (PDB) entry 5II0] and confirmed with data from mutagenesis. (9) For the CGRP receptor, a crystal structure is available showing the C-terminal tail of a CGRP analogue bound to the ECDs of CLR and RAMP1. (10) Extensive mutagenesis data that have allowed the construction of models of how CGRP interacts with the TM domain of the receptor are available. (11−15) These studies have implicated extracellular loop 2 (ECL2) of CLR as being especially important for CGRP binding. (12−14) However, direct evidence of molecular contacts between the ligand and receptor is lacking.
Photoaffinity cross-linking has long been used to map the binding of ligands to receptors. (16) Conventionally, a photoaffinity cross-linker is incorporated into the ligand; this then requires extensive peptide mapping to identify the most likely contact point on the receptor. With the use of genetic code expansion and unnatural amino acid mutagenesis, (17) it is now possible to incorporate the photoaffinity cross-linker into the receptor, thereby eliminating the need for peptide mapping or sequencing to identify contact points on the receptor. This general approach, known as targeted photo-cross-linking, (18) has been used to map ligand–receptor interactions in a number of GPCRs, including the neurokinin NK-1 receptor, (19) ghrelin receptor, (20) type 1 receptor for the corticotropin releasing factor (CRF1R), (21−23) and GLP-1 receptor. (24) This has not yet been extensively applied to a complex dimeric receptor, such as the CGRP receptor. In the study presented here, we have developed a modified targeted photo-cross-linking strategy to study ECL2 of CLR and have identified two major contact points for CGRP: I284 and L291.

Materials and Methods

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Materials

Human α CGRP was from Bachem (St. Helens, U.K.). CGRP analogues were synthesized as indicated below. Alpha Screen, LANCE cAMP assay kits, reagents, plates, and 125I-labeled human iodohistidyl10-α-CGRP were purchased from PerkinElmer (Waltham, MA).

Peptide Synthesis

[Lys(5(6)-carboxyfluorescein)15]-α-CGRP, [Leu12]-α-CGRP, and [Leu16]-α-CGRP were synthesized by solid phase peptide synthesis using the Fmoc/tBu method on a 0.1 mmol scale largely as described previously. (25) Briefly, Rink amide ChemMatrix resin (0.48 mmol/g) was elongated using a PS3 (Protein Technologies, Tucson, AZ) automated peptide synthesizer. The Fmoc group was removed with 20% piperidine in dimethylformamide (DMF) (v/v) for 2 × 5 min, and the Fmoc amino acids were coupled for 20 min using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate and N,N-diisopropylethylamine. A pseudoproline, Fmoc-Leu-Ser[ψ-Me,MePro] was used at position 16/17 to increase the synthetic efficiency. (26) To enable incorporation of the 5(6)-carboxyfluorescein to a side chain amine, Leu15 was substituted with Lys(Dde) [Dde = 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl]. Boc-alanine was used as the N-terminal amino acid.
Following chain assembly, on-resin removal of the Dde group on Lys15 was accomplished with 2% hydrazine hydrate in DMF (v/v) for 2 × 3 min, and 5(6)-carboxyfluorescein was coupled with N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole overnight. The resin was then treated with 20% piperidine in DMF (v/v) for 4 × 5 min to remove excess 5(6)-carboxyfluorescein. (27) The crude peptide was cleaved from the resin with concomitant removal of side chain protecting groups with 94% trifluoroacetic acid, 1% triisopropylsilane, 2.5% water, and 2.5% ethanedithiol (v/v/v/v) for 3 h, precipitated with cold diethyl ether, isolated by centrifugation, dissolved in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid, and lyophilized to afford 228 mg of the crude peptide. The crude fluorescently labeled α-CGRP was analyzed by liquid chromatography and mass spectrometry (calcd mass of 4165.33 Da, found mass of 4163.30 ± 0.9 Da) and then purified by sample displacement chromatography (28) on a C18 Gemini (Phenomenex) column (10 mm × 250 mm) at a flow rate of 5 mL/min at 50 °C to give 25.1 mg of pure linear (unoxidized) [Lys(5(6)-carboxyfluorescein)15]-α-CGRP.
The linear peptide (25 mg) was dissolved in 0.1 M Tris-HCl (pH 7.85) at a concentration of 1 mg/mL, and the oxidation (disulfide formation) was allowed to proceed at room temperature open to air. Monitoring by reverse phase high-performance liquid chromatography (HPLC) indicated that the reaction was complete after 5 h. The reaction mixture was acidified to pH 2, diluted with water (60 mL), and purified by semipreparative reverse phase HPLC using a C18 Gemini column (10 mm × 250 mm) at a flow rate of 5 mL/min at 50 °C and eluted using an appropriate gradient based on the analytical HPLC profile. Fractions containing the pure peptide were identified by electrospray mass spectrometry and/or HPLC, pooled, and lyophilized to give [Lys(5(6)-carboxyfluorescein)15]-α-CGRP at >95% purity as judged by integration of the HPLC chromatogram at 210 nm (calcd mass of 4163.31 Da, found mass of 4162.20 ± 0.5 Da).

Unnatural Amino Acid Mutagenesis and Cross-Linking Strategy

Unnatural amino acid mutagenesis was performed in HEK293T cells as described previously, (24,29) using Amber mutant human CLR, myc-tagged RAMP1, suppressor tRNA, and azF aminoacyl tRNA synthetase but with some modifications. HEK293T cells were seeded to a density of 800000 cells per well (resulting in 70% cell confluence the next day) of a six-well plate. Twenty-four hours after being seeded, the cells were transfected with a DNA cocktail comprising 1 μg of mutated/WT receptor, 1 μg of suppressor tRNA, and 0.5 μg of aminoacyl tRNA synthetase using lipofectamine (Life Technologies catalog no. 18324-012) as per the manufacturer’s instructions. Four hours post-transfection, the medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% (v/v) FBS and 1 mM azF, which itself was subsequently replaced after 24 h with DMEM supplemented with 20% (v/v) FBS and 0.5 mM azF. Cells were harvested after a further 24 h, and the membrane fraction was isolated by centrifugation. The membranes were incubated with the labeled peptide for 1 h at room temperature. Once the incubation was complete, the sample was split into two equal parts. One part was pipetted into a quartz cuvette and exposed to 254 nm light for 1 h (an exposure longer than that previously reported (24)), while the other was wrapped in aluminum foil and also placed in the light source. Heat was dissipated in both samples using silicone tubing inserted into the cuvette that was flushed with ice-cold water. Cross-linking was performed in a UVP cross-linker (Analytik-Jena AG) at an energy of 100 μJ/cm, (2) with the sample approximately 10 cm from the light source. Both the cross-linked samples and the control samples were pelleted by centrifugation, washed using phosphate-buffered saline, and solubilized using dodecyl maltoside for 4 h. Post-solubilization, the samples were centrifuged at 30000g for 30 min, after which 150 μL of the soluble fraction was pipetted into a well of a 96-well plate and read using a Mithras LB 940 instrument (Berthould). The cross-linked and control samples were compared to determine the degree of peptide labeling.

Tyrosine Mutagenesis

Individual amino acids in human HA-tagged CLR were mutated to tyrosine as described previously. (11,12,14)

cAMP Production

Human HA-tagged CLR (WT or mutant) and myc-tagged RAMP1 were transfected into HEK293T or HEK293S cells, stimulated with human α-CGRP or synthetic peptides, and cAMP accumulation was measured via Alpha Screen or LANCE as previously described. (11,13,30)

Radioligand Binding

Competition radioligand binding assays were performed on HEK293S membranes using 125I-labeled human α-CGRP. HEK293S cells were grown in 15 cm dishes, and each dish was transfected with 30 μg of human HA-tagged CLR and 30 μg of myc-tagged RAMP1 using polyethylenimine (PEI) as described previously. (13) Forty-eight hours after transfection, membranes were isolated by homogenizing cells followed by centrifugation at 100000g for 1 h at 4 °C, and membranes were resuspended and stored at −80 °C. The binding assay was performed essentially as described previously (31) except the membranes were incubated with labeled and unlabeled peptides for 1 h at room temperature.

Data Analysis

Concentration response curves were fitted to a three-parameter logistic equation to obtain Emax, pEC50, and basal responses. The Hill coefficient was constrained to unity. Curve fitting was performed with GraphPad Prism version 7, as described previously. (13) For photoaffinity cross-linking, paired Student’s t tests were used to assess if the total cross-linking for each mutant was statistically different from the nonspecific cross-linking, accepting significance at a P < 0.05 level.

Molecular Modeling

Homology models of the active CLR were generated using Modeler (32) utilizing the recently determined cryo-electron microscope structures of the activated GLP-1 and calcitonin receptors (PDB entries 5VAI and 5UZ7, respectively). (8,9) One thousand models combining homology-based restraints from both templates (>2 Å root-mean-square deviation between models) were generated by Modeler using the default method. The transmembrane and loop regions of the Modeler-based structures were further refined and ranked using the membrane relax module of ROSETTA. (33) The refined models were clustered, and the best model was chosen on the basis of the largest cluster with the lowest ROSETTA membrane score.
The peptide was initially ab initio folded using an in-house script with a disulfide bond constraining Cys2 and Cys7 of the peptide. Regions of secondary structure (34) were also imposed on the ab initio simulation. The best scoring folded peptide was docked on the basis of the FlexPepDock module of ROSETTA; 10000 docking solutions between the peptide and receptor were generated. The FlexPepDock docked solutions were filtered in two stages. The docked solutions were initially filtered using analogous contact restraints (cutoff of 5 Å) as observed in the 4RWG crystal structure. (9) These results were further filtered by keeping any solution in which both Ile284 and Leu291 were within 5 Å of any atom of the docked CGRP ligand. The filtered results (737 docked poses) were then further refined using the membrane relax module also found in the ROSETTA suite of software. The middle structure of the largest, lowest-energy cluster was chosen for further analysis.
Replica exchange Monte Carlo simulations as implemented in Hippo (35) were used explore the conformational space of the ROSETTA-refined model. Sixteen replicas were simulated over the temperature range of 300–500 K following a Boltzmann distribution in a 30 Å implicit membrane. Other variables were kept as the default settings. The trajectory was visualized using Visual Molecular Dynamics. (36)

Results

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Activity of [Lys(5(6)-carboxyfluorescein)15] Human α-CGRP (15-Fluo CGRP)

To identify cross-linking to CLR, a fluorescent CGRP agonist was required. On the basis of previously published structure–activity data, (37) supported by molecular modeling, (11,38) we reasoned that it should be possible to modify CGRP at position 15 while retaining agonist activity. The native Leu at this residue was replaced by Lys and derivatized with 5(6)-carboxyfluorescein. The resulting analogue was evaluated for its ability to stimulate cAMP production via the CGRP receptor. While it had reduced potency, it was a full agonist (Figure 1), suggesting that this modification did not greatly affect the functional properties of the ligand

Figure 1

Figure 1. cAMP production on HEK293T cells transfected with CLR and RAMP1 and exposed to CGRP or 15-Fluo CGRP for 15 min. Values are means ± the standard error of the mean (SEM); n = 3 independent experiments.

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Confirmation of the Cross-Linking Strategy

Preliminary experiments indicated that optimal specific cross-linking was obtained following a 1 h exposure to ultraviolet (UV) light. The availability of a crystal structure showing the binding of a modified CGRP analogue to the extracellular domain of the CGRP receptor (10) provided a system for evaluating our methodology. A series of azF mutants was constructed in which residues within 5 Å of or more than 7 Å from the bound CGRP analogue were substituted. There was excellent agreement between the ability of the mutants to cross-link to CGRP and their distances from the bound CGRP analogue in the crystal structure (Figure 2).

Figure 2

Figure 2. Fluorescence of solubilized HEK293T cell membranes from cells transfected with RAMP1 and CLR containing azF substitutions at the indicated residues. Membranes were exposed to 15-Fluo CGRP in the presence or absence of UV light. D94, H114, W121, and N128 are all within 5 Å of bound CGRP; (10) other residues are 7–10 Å away. Values are means ± SEM; n = 3. *P < 0.05, and **P < 0.01; values of total labeling that are significantly different from the nonspecific values in the paired control samples, i.e., in the absence of UV light.

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Cross-Linking by Residues in ECL2

A scan was then performed on ECL2 of CLR and its flanking regions from H270 to Y292, excluding C282, which is involved in a conserved disulfide bond with C212 at the extracellular end of TM3. The data suggest that major points of attachment for 15-Fluo CGRP are I284 and L291, with much weaker although significant labeling in a number of other positions at the distal end of the loop (Figure 3). There was virtually no specific labeling at any position in the loop proximal to D280. It is possible that a lack of labeling might simply reflect the fact that the receptor is nonfunctional when substituted with azF. To test this, a Tyr scan of the residues mutated to azF was performed, excluding the native tyrosines at positions 277 and 278 (Figure 4). A Tyr rather than an azF scan was performed because the modified aminoacyl tRNA synthetase used to incorporate azF will also accept Tyr, albeit at lower efficiency, resulting in a mixed population of receptors potentially with one of two different residues at the same site. (24) Tyr is structurally the closest natural amino acid to azF. The results showed that all the mutants assessed should still be able to interact with 15-Fluo CGRP at 100 nM, the concentration used in our cross-linking experiments, as the Tyr mutants retained a good sensitivity to CGRP.

Figure 3

Figure 3. Fluorescence of solubilized HEK293T cell membranes from cells transfected with RAMP1 and CLR containing azF substitutions at the indicated residues. Membranes were exposed to 15-Fluo CGRP in the presence or absence of UV light. The values are the specific labeling, following subtraction of the nonspecific from total bound fluorescence. Values are means ± SEM; n = 3–6. *P < 0.05; values for which total labeling is significantly different from the nonspecific values in the paired control samples, i.e., in the absence of UV light. Nonspecific labeling ranged from 2247 to 5759 AU.

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Figure 4

Figure 4. Tyrosine scan of ECL2. Potency of human α-CGRP at stimulating cAMP production in HEK293T cells transfected with CLR (WT or mutants) and RAMP1. Values are means ± SEM; n = 3.

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Role of TM3 and TM6

The current cross-linking data and previous mutation studies have indicated that ECL2 and the first turn of TM5 are important in forming the binding site for CGRP. We extended the use of our cross-linking method to address the role of other parts of the receptor. M223 (TM3) and F349 (TM6) were mutated to azF. These residues are likely to face into the TM bundle of CLR but are approximately two helical turns deeper into the TM bundle than L291 is (Figure 6). Thus, they provide insight into how far CGRP penetrates into the TM core of CLR. The specific cross-linking seen with both of these residues was comparable with that of S286 (Figure 5), which appears to act as a minor contact in ECL2 (Figure 3), although we cannot exclude a lower cross-linking efficiency due to poor photolysis of residues deep in the TM bundle. It seems likely that M223 and F349 are at the base of the binding pocket for CGRP.

Figure 5

Figure 5. Fluorescence of solubilized HEK293T cell membranes from cells transfected with RAMP1 and CLR containing azF substitutions at the indicated residues. Membranes were exposed to 15-Fluo CGRP in the presence or absence of UV light. Values are means ± SEM; n = 3. *P < 0.05; values of total labeling significantly different from the nonspecific values in the paired control samples, i.e., in the absence of UV light.

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Molecular Modeling of Bound CGRP

Our modeling suggests that CGRP extends down as far as H295, in TM5. I284 is close to Leu12 and Ala13 of CGRP, and L291 is adjacent to Thr6 of CGRP (Figure 6). Leu16 of CGRP is the most proximal residue of the bound CGRP to make potential contact with the ECLs. It is close to I284 and V198 (in ECL1) but is also in the proximity of I32 of the ECD, perhaps making contact with both ECLs and the ECD of CLR.

Figure 6

Figure 6. Molecular model of the CLR (brown ribbon)/RAMP1 (blue ribbon; TM region omitted for the sake of clarity) complex with bound CGRP (pink ribbon). (A) Detail of predicted interactions of CGRP with I284 and L291. (B) Predicted position of bound CGRP with residues on the periphery of the binding site. CLR residues shown in space-filling mode; CGRP residues colored green. The models can be viewed online using Molecule Viewer (39) at https://bit.ly/2FnQAHp (recommended browsers are Google Chrome in incognito mode or Firefox). (C) Full length structure of the CLR complex. The positions of TM5 and helix 8 are highlighted to aid orientation.

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Activity of [Leu12Ala]- and [Leu16Ala]-Human α-CGRP

To further test our proposed mode of binding, we investigated the ability of [Leu12Ala]- and [Leu16Ala]-human α-CGRP to stimulate cAMP. Both analogues showed substantial reductions in potency and Emax, with [Leu16Ala]-human α-CGRP showing the largest effects (Figure 7). This result was confirmed in a radioligand binding assay, in which [Leu16Ala]-human α-CGRP also showed a substantial reduction in affinity.

Figure 7

Figure 7. Comparison of the ability of [Leu12Ala]- and [Leu16Ala]-human α-CGRP (A) to stimulate cAMP in HEK293S cells transfected with HA-CLR and myc-RAMP1 or (B) to displace [125I]CGRP at the CGRP receptor. Values are means ± SEM; n = 3 or 4.

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Discussion

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We have used unnatural amino acid mutagenesis to map key residues in and adjacent to ECL2 of CLR that contribute to the binding of CGRP. The data suggest that I284 and L291 make contacts in the vicinity of Thr6 and Leu12 of bound CGRP. This binding orientation is consistent with the modeled position of calcitonin in the cryo-electron microscopy structure of the calcitonin receptor. (9)
A number of the residues that we have identified in this study as contributing to the binding of CGRP have been previously identified as being important for CGRP production of cAMP, based on mutagenesis. We predicted that I284 could interact with one or more CGRP residues C-terminal to the 2–7 disulfide-bonded loop of the peptide, conventionally considered to be the mediator of receptor activation. (13) However, using an alanine scan, we have previously found that changes to Val8 and Thr9 of CGRP can alter its efficacy. (25) Our current model suggests that Val8 is adjacent to H194 in TM2 and Thr9 is in the proximity of the backbone of ECL2. Our study further extends this by demonstrating that Leu16 in CGRP can influence efficacy, potentially by contacting I32 in the ECD of CLR. Furthermore, we have observed during a replica exchange Monte Carlo simulation of the bound CGRP (to investigate dynamic stability), L16 periodically interacts with V198 (ECL1) and I284 of CLR. V198 and I32 have both been shown to be important for cAMP production by CGRP. (40,41) These observations suggest how CGRP residues outside of the classic 1–7 “activation” domain of the peptide can influence efficacy; we speculate that Leu16 may be particularly important in interacting with both the ECLs and ECD of CLR. It may therefore be possible to develop truncated analogues that retain agonist activity by interacting with the upper regions of the TM bundle and ECLs, as has been done for GLP-1. (42)
Within the 1–7 region of CGRP, our previous structure–activity work indicated that Thr6 was essential for agonism. In fact, this residue is absolutely conserved in all members of the calcitonin family of peptides. (25,34) In our model, Thr6 of CGRP is close to not only L291 of TM5 but also H295 a turn lower. Previous mutagenesis has shown that H295 is important for the activation of all CLR/RAMP complexes with both CGRP and adrenomedullin; (11) its equivalent is also important for agonism by calcitonin in the calcitonin receptor. (9) Molecular dynamics simulations indicated that H295, at the top of TM5, controls interhelical contacts among TM3, TM5, TM6, and TM7 of CLR. A rotation of this residue caused helix sliding and tilting that opened the G protein binding pocket. (11) Residues deeper in the receptor that are important for receptor activation such as E348 (11) would seem unlikely to be significant ligand contacts, unless they are connected by bound water molecules.
Although I284 and L291 were the main contacts revealed in this study, it is interesting to note that there were a range of minor contacts in the distal part of ECL2. The pattern here was that every other residue between I284 and L290 appeared as contacts. Interestingly, our previous mutagenesis and modeling have suggested that this forms a short stretch of β-strand, along the rim of the cavity between TM4 and TM5. (12) This secondary structure element is consistent with the labeling pattern.
In eight of 15 of the human family B GPCRs, the equivalent of I284 is glutamic or aspartic acid; in three additional receptors, it is threonine. Only in CLR, the calcitonin receptor (leucine), and the corticotrophin receptors (phenylalanine) is the corresponding residue hydrophobic. Thus, within family B GPCRs, this residue may be important in ligand specificity; it faces into the core of the receptor where it can be resolved in structures. In the calcitonin receptor, the equivalent residue is important for the binding of human and porcine but not salmon calcitonin, indicating how similar ligands can differentially interact with residues within their binding pockets. (43) The absence of labeling for W283 is noteworthy as this residue is highly conserved in family B GPCRs and mutation may show deleterious effects. (44,45) Its side chain orientation is variable in the current family B GPCR structures, either facing into the core in the calcitonin and glucagon receptor structures or inserting between TM3 and TM4 in other structures. (4,5,9,46) These data suggest that W283 does not contact the peptide, indicating it is most likely to be inserted between TM3 and TM4 (Figure 6B).
The techniques described in this paper may be applied to other receptors. While previously the method has been applied to the GLP-1 receptor, (24) our experience is that significant refinement is required. In the study presented here, it was necessary to illuminate the receptor for 1 h to obtain cross-linking, rather than for the 5 min used in the GLP-1 study. This difference may reflect the fact that the GLP-1 study was focused on the extracellular domain of the receptor; greater exposure times may be necessary for residues within the membrane domain. In studies of CRF1R, where transmembrane residues were also targeted, illumination was performed for 20–40 min. (21,22) Thus, investigators should optimize labeling conditions to ensure their protocols are maximally effective. The cross-linking efficiency will also depend on the power of the UV source and the distance of the sample from the light, which are likely to vary between laboratories.
In conclusion, this study suggests that CGRP binds to a pocket formed by the ECLs of CLR, penetrating approximately two turns into the TM region. Direct contacts with residues here and in the ECLs form the basis of the agonist-induced conformational shift in CLR during receptor activation.

Author Information

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  • Corresponding Author
  • Authors
    • John Simms - Aston University, Birmingham B4 7ET, U.K.;  Coventry University, Priory Street, Coventry CV1 5FB, U.K.
    • Romez Uddin - Aston University, Birmingham B4 7ET, U.K.
    • Thomas P. Sakmar - The Rockefeller University, 1230 York Avenue, New York, New York 10065, United States
    • Joseph J. Gingell - University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
    • Michael L. Garelja - University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
    • Debbie L. Hay - University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand;  Orcidhttp://orcid.org/0000-0002-9558-5122
    • Margaret A. Brimble - University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand;  Orcidhttp://orcid.org/0000-0002-7086-4096
    • Paul W. Harris - University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
    • Christopher A. Reynolds - University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
  • Funding

    This work was supported by BBSRC grants to D.R.P. (BB/M007529/1) and C.A.R. (BB/M006883/1). C.A.R. is a Royal Society Industry Fellow. D.L.H. is a James Cook Research Fellow of the Royal Society of New Zealand.

  • Notes
    The authors declare no competing financial interest.

    To access the research data supporting this publication, see https://doi.org/10.17036/researchdata.aston.ac.uk.00000368.

Acknowledgments

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The authors thank Dr. Casandra Koole and Dr. Harriet Watkins for advice. Furthermore, the authors thank Merry Wang at Autodesk for help and advice with Molecule Viewer.

Abbreviations

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azF-p

azido l-phenylalanine

AM

adrenomedullin

CGRP

calcitonin gene-related peptide

CLR

calcitonin receptor-like receptor

CT

calcitonin

Dde

1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl

DMF

dimethylformamide

ECD

extracellular domain

ECL

extracellular loop

GLP-1

glucagon-like peptide-1

GPCR

G protein-coupled receptor

RAMP

receptor activity-modifying protein

TM

transmembrane.

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This article references 46 other publications.

  1. 1
    Hay, D. L., Garelja, M. L., Poyner, D. R., and Walker, C. S. (2018) Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25. Br. J. Pharmacol. 175, 3– 17,  DOI: 10.1111/bph.14075
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    1
    Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25
    Hay, Debbie L.; Garelja, Michael L.; Poyner, David R.; Walker, Christopher S.
    British Journal of Pharmacology (2018), 175 (1), 3-17CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
    The calcitonin/CGRP family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two GPCRs, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerization of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM1 and AM2 receptors (CLR/RAMP2 or RAMP3) and the AMY1, AMY2 and AMY3 receptors (CTR/RAMPs1-3 complexes, resp.). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors, and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT(a) and CT(b)). Furthermore, the AMY1(a) receptor is activated equally well by both amylin and CGRP, and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example, many agents targeting the CGRP system are in clin. trials, and pramlintide, an amylin analog, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacol. of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clin. Pharmacol. and colleagues.
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    Hay, D. L. and Walker, C. S. (2017) CGRP and its receptors. Headache 57, 625– 636,  DOI: 10.1111/head.13064
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    CGRP and its receptors
    Hay Debbie L; Walker Christopher S
    Headache (2017), 57 (4), 625-636 ISSN:.
    The calcitonin gene-related peptide (CGRP) neuropeptide system is an important but still evolving target for migraine. A fundamental consideration for all of the current drugs in clinical trials and for ongoing development in this area is the identity, expression pattern, and function of CGRP receptors because this knowledge informs safety and efficacy considerations. In recent years, only the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (RAMP1) complex, known as the CGRP receptor, has generally been considered relevant. However, CGRP is capable of activating multiple receptors and could have more than one endogenous receptor. The recent identification of the CGRP-responsive calcitonin receptor/RAMP1 complex (AMY1 receptor - amylin subtype 1 receptor) in the trigeminovascular system warrants a deeper consideration of the molecular identity of CGRP receptor(s) involved in the pathophysiology, and thus potential treatment of migraine. This perspective considers some of the issues and implications.
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  3. 3
    Hoare, S. R. (2005) Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors.. Drug Discovery Today 10, 417– 427,  DOI: 10.1016/S1359-6446(05)03370-2
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    Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors
    Hoare, Sam R. J.
    Drug Discovery Today (2005), 10 (6), 417-427CODEN: DDTOFS; ISSN:1359-6446. (Elsevier)
    A review. Class B G-protein-coupled receptors are a small family of 15 peptide-binding receptors. This family includes at least six biol. attractive therapeutic targets for both peptide ligands (osteoporosis and Type II diabetes) and nonpeptide ligands (anxiety, depression and migraine). A general mechanism of peptide binding has emerged for this receptor family, termed the two-domain model. In this mechanism, the C-terminal ligand region binds the extracellular N-terminal domain of the receptor. This interaction acts as an affinity trap, promoting interaction of the N-terminal ligand region with the juxtamembrane domain of the receptor. Peptide binding to the juxtamembrane domain activates the receptor and stimulates intracellular signaling. Nonpeptide ligands bind the juxtamembrane or N-terminal domain and, in most cases, allosterically modulate peptide-ligand binding. Here, these mechanisms of peptide and nonpeptide ligand binding are reviewed, then applied in a discussion of the future strategies of drug development for Class B G-protein-coupled receptors.
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  4. 4
    Zhang, H., Qiao, A., Yang, D., Yang, L., Dai, A., de Graaf, C., Reedtz-Runge, S., Dharmarajan, V., Zhang, H., Han, G. W., Grant, T. D., Sierra, R. G., Weierstall, U., Nelson, G., Liu, W., Wu, Y., Ma, L., Cai, X., Lin, G., Wu, X., Geng, Z., Dong, Y., Song, G., Griffin, P. R., Lau, J., Cherezov, V., Yang, H., Hanson, M. A., Stevens, R. C., Zhao, Q., Jiang, H., Wang, M. W., and Wu, B. (2017) Structure of the full-length glucagon class B G-protein-coupled receptor. Nature 546, 259– 264,  DOI: 10.1038/nature22363
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    Structure of the full-length glucagon class B G-protein-coupled receptor
    Zhang, Haonan; Qiao, Anna; Yang, Dehua; Yang, Linlin; Dai, Antao; de Graaf, Chris; Reedtz-Runge, Steffen; Dharmarajan, Venkatasubramanian; Zhang, Hui; Han, Gye Won; Grant, Thomas D.; Sierra, Raymond G.; Weierstall, Uwe; Nelson, Garrett; Liu, Wei; Wu, Yanhong; Ma, Limin; Cai, Xiaoqing; Lin, Guangyao; Wu, Xiaoai; Geng, Zhi; Dong, Yuhui; Song, Gaojie; Griffin, Patrick R.; Lau, Jesper; Cherezov, Vadim; Yang, Huaiyu; Hanson, Michael A.; Stevens, Raymond C.; Zhao, Qiang; Jiang, Hualiang; Wang, Ming-Wei; Wu, Beili
    Nature (London, United Kingdom) (2017), 546 (7657), 259-264CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiol. of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR contg. both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as obsd. in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen-deuterium exchange, disulfide crosslinking and mol. dynamics studies suggest that the stalk and the first extracellular loop have crit. roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.
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  5. 5
    Jazayeri, A., Rappas, M., Brown, A. J. H., Kean, J., Errey, J. C., Robertson, N. J., Fiez-Vandal, C., Andrews, S. P., Congreve, M., Bortolato, A., Mason, J. S., Baig, A. H., Teobald, I., Dore, A. S., Weir, M., Cooke, R. M., and Marshall, F. H. (2017) Crystal structure of the GLP-1 receptor bound to a peptide agonist. Nature 546, 254– 258,  DOI: 10.1038/nature22800
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    Crystal structure of the GLP-1 receptor bound to a peptide agonist
    Jazayeri, Ali; Rappas, Mathieu; Brown, Alastair J. H.; Kean, James; Errey, James C.; Robertson, Nathan J.; Fiez-Vandal, Cedric; Andrews, Stephen P.; Congreve, Miles; Bortolato, Andrea; Mason, Jonathan S.; Baig, Asma H.; Teobald, Iryna; Dore, Andrew S.; Weir, Malcolm; Cooke, Robert M.; Marshall, Fiona H.
    Nature (London, United Kingdom) (2017), 546 (7657), 254-258CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the mol. mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helixes reveals hallmarks of an active conformation similar to that obsd. in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
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  6. 6
    Zhang, H., Qiao, A., Yang, L., Van Eps, N., Frederiksen, K. S., Yang, D., Dai, A., Cai, X., Zhang, H., Yi, C., Cao, C., He, L., Yang, H., Lau, J., Ernst, O. P., Hanson, M. A., Stevens, R. C., Wang, M. W., Reedtz-Runge, S., Jiang, H., Zhao, Q., and Wu, B. (2018) Structure of the glucagon receptor in complex with a glucagon analogue. Nature 553, 106– 110,  DOI: 10.1038/nature25153
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    6
    Structure of the glucagon receptor in complex with a glucagon analogue
    Zhang, Haonan; Qiao, Anna; Yang, Linlin; Van Eps, Ned; Frederiksen, Klaus S.; Yang, Dehua; Dai, Antao; Cai, Xiaoqing; Zhang, Hui; Yi, Cuiying; Cao, Can; He, Lingli; Yang, Huaiyu; Lau, Jesper; Ernst, Oliver P.; Hanson, Michael A.; Stevens, Raymond C.; Wang, Ming-Wei; Reedtz-Runge, Steffen; Jiang, Hualiang; Zhao, Qiang; Wu, Beili
    Nature (London, United Kingdom) (2018), 553 (7686), 106-110CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resoln. limitations, the specific mol. interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding interdomain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resoln. crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analog and partial agonist, NNC1702. This structure provides mol. details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual binding-site trigger model for GCGR activation - which requires conformational changes of the stalk, first extracellular loop and TMD - that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.
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  7. 7
    Liang, Y. L., Khoshouei, M., Glukhova, A., Furness, S. G. B., Zhao, P., Clydesdale, L., Koole, C., Truong, T. T., Thal, D. M., Lei, S., Radjainia, M., Danev, R., Baumeister, W., Wang, M. W., Miller, L. J., Christopoulos, A., Sexton, P. M., and Wootten, D. (2018) Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121– 125,  DOI: 10.1038/nature25773
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    Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex
    Liang, Yi-Lynn; Khoshouei, Maryam; Glukhova, Alisa; Furness, Sebastian G. B.; Zhao, Peishen; Clydesdale, Lachlan; Koole, Cassandra; Truong, Tin T.; Thal, David M.; Lei, Saifei; Radjainia, Mazdak; Danev, Radostin; Baumeister, Wolfgang; Wang, Ming-Wei; Miller, Laurence J.; Christopoulos, Arthur; Sexton, Patrick M.; Wootten, Denise
    Nature (London, United Kingdom) (2018), 555 (7694), 121-125CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gαs heterotrimer, detd. at a global resoln. of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure. At the intracellular face, there was a six-degree difference in the angle of the Gαs-α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addn., the structures differed in the rate and extent of conformational reorganization of the Gαs protein. Our structure provides insights into the mol. basis of biased agonism.
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  8. 8
    Zhang, Y., Sun, B., Feng, D., Hu, H., Chu, M., Qu, Q., Tarrasch, J. T., Li, S., Sun Kobilka, T., Kobilka, B. K., and Skiniotis, G. (2017) Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248– 253,  DOI: 10.1038/nature22394
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    Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein
    Zhang, Yan; Sun, Bingfa; Feng, Dan; Hu, Hongli; Chu, Matthew; Qu, Qianhui; Tarrasch, Jeffrey T.; Li, Shane; Sun Kobilka, Tong; Kobilka, Brian K.; Skiniotis, Georgios
    Nature (London, United Kingdom) (2017), 546 (7657), 248-253CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metab. and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near at. resoln. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5-helix of the Ras-like domain of Gs. These results provide a structural framework for understanding class B GPCR activation through hormone binding.
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    Liang, Y. L., Khoshouei, M., Radjainia, M., Zhang, Y., Glukhova, A., Tarrasch, J., Thal, D. M., Furness, S. G. B., Christopoulos, G., Coudrat, T., Danev, R., Baumeister, W., Miller, L. J., Christopoulos, A., Kobilka, B. K., Wootten, D., Skiniotis, G., and Sexton, P. M. (2017) Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118– 123,  DOI: 10.1038/nature22327
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    Phase-plate cryo-EM structure of a class B GPCR-G-protein complex
    Liang, Yi-Lynn; Khoshouei, Maryam; Radjainia, Mazdak; Zhang, Yan; Glukhova, Alisa; Tarrasch, Jeffrey; Thal, David M.; Furness, Sebastian G. B.; Christopoulos, George; Coudrat, Thomas; Danev, Radostin; Baumeister, Wolfgang; Miller, Laurence J.; Christopoulos, Arthur; Kobilka, Brian K.; Wootten, Denise; Skiniotis, Georgios; Sexton, Patrick M.
    Nature (London, United Kingdom) (2017), 546 (7656), 118-123CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Class B G-protein-coupled receptors (GPCRs) are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes, and obesity. Here, we report the structure of a full-length class B GPCR receptor, the calcitonin receptor, in complex with the peptide ligand, calcitonin, and a heterotrimeric Gαsβγ protein detd. by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist, calcitonin, engaged the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helixes 6 and 7. This conformation was accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also obsd. was an extended intracellular helix 8 that contributed to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. Thus, this structure provides a new framework for understanding GPCR receptor function.
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    Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor. Mol. Cell 58, 1040– 1052,  DOI: 10.1016/j.molcel.2015.04.018
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    Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor
    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.
    Molecular Cell (2015), 58 (6), 1040-1052CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Assocn. of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolns., resp. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacol. data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.
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    Woolley, M. J., Reynolds, C. A., Simms, J., Walker, C. S., Mobarec, J. C., Garelja, M. L., Conner, A. C., Poyner, D. R., and Hay, D. L. (2017) Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs. Biochem. Pharmacol. 142, 96– 110,  DOI: 10.1016/j.bcp.2017.07.005
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    11
    Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs
    Woolley, Michael J.; Reynolds, Christopher A.; Simms, John; Walker, Christopher S.; Mobarec, Juan Carlos; Garelja, Michael L.; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.
    Biochemical Pharmacology (Amsterdam, Netherlands) (2017), 142 (), 96-110CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
    Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP prodn., modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting mol. dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The mol. dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.
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    Woolley, M. J., Simms, J., Mobarec, J. C., Reynolds, C. A., Poyner, D. R., and Conner, A. C. (2017) Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach. Mol. Cell. Endocrinol. 454, 39– 49,  DOI: 10.1016/j.mce.2017.05.034
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    Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach
    Woolley, Michael J.; Simms, John; Mobarec, Juan Carlos; Reynolds, Christopher A.; Poyner, David R.; Conner, Alex C.
    Molecular and Cellular Endocrinology (2017), 454 (), 39-49CODEN: MCEND6; ISSN:0303-7207. (Elsevier Ireland Ltd.)
    The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution anal. method and mol. modeling to the functionally important residues of ECL2 to reveal key mol. interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest redn. in signalling following alanine substitution anal. and comprise a group of basic, acidic and arom. residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochem. requirements for neighboring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.
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    Woolley, M. J., Simms, J., Uddin, S., Poyner, D. R., and Conner, A. C. (2017) Relative Antagonism of Mutants of the CGRP Receptor Extracellular Loop 2 Domain (ECL2) Using a Truncated Competitive Antagonist (CGRP8–37): Evidence for the Dual Involvement of ECL2 in the Two-Domain Binding Model. Biochemistry 56, 3877– 3880,  DOI: 10.1021/acs.biochem.7b00077
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    Woolley, M. J., Watkins, H. A., Taddese, B., Karakullukcu, Z. G., Barwell, J., Smith, K. J., Hay, D. L., Poyner, D. R., Reynolds, C. A., and Conner, A. C. (2013) The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach,. J. R. Soc., Interface 10, 20130589,  DOI: 10.1098/rsif.2013.0589
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    14
    The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach
    Woolley, Michael J.; Watkins, Harriet A.; Taddese, Bruck; Karakullukcu, Z. Gamze; Barwell, James; Smith, Kevin J.; Hay, Debbie L.; Poyner, David R.; Reynolds, Christopher A.; Conner, Alex C.
    Journal of the Royal Society, Interface (2013), 10 (88), 20130589/1-20130589/11CODEN: JRSICU; ISSN:1742-5689. (Royal Society)
    The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modeling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.
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  15. 15
    Vohra, S., Taddese, B., Conner, A. C., Poyner, D. R., Hay, D. L., Barwell, J., Reeves, P. J., Upton, G. J., and Reynolds, C. A. (2013) Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies, Journal of the Royal Society. J. R. Soc., Interface 10, 20120846,  DOI: 10.1098/rsif.2012.0846
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    15
    Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies
    Vohra, Shabana; Taddese, Bruck; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.; Barwell, James; Reeves, Philip J.; Upton, Graham J. G.; Reynolds, Christopher A.
    Journal of the Royal Society, Interface (2013), 10 (79), 20120846/1-20120846/13CODEN: JRSICU; ISSN:1742-5689. (Royal Society)
    Modeling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homol. The plant GPCR, GCR1, has homol. to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating max. lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this anal. to generate active and inactive homol. models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the anal. of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to assoc. the key mol. features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg2.39, His2.43 and Glu3.46, which makes a polar lock with T6.37. These alignments and models provide useful tools for understanding class B GPCR function.
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  16. 16
    Dong, M. and Miller, L. J. (2006) Use of photoaffinity labeling to understand the molecular basis of ligand binding to the secretin receptor. Ann. N. Y. Acad. Sci. 1070, 248– 264,  DOI: 10.1196/annals.1317.023
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    Use of photoaffinity labeling to understand the molecular basis of ligand binding to the secretin receptor
    Dong, Maoqing; Miller, Laurence J.
    Annals of the New York Academy of Sciences (2006), 1070 (VIP, PACAP, and Related Peptides), 248-264CODEN: ANYAA9; ISSN:0077-8923. (Blackwell Publishing, Inc.)
    A review. The secretin receptor was the first member of the Class B family of G protein-coupled receptors that was identified in 1991, 89 years after secretin action was first recognized. That report resulted in the introduction of the term hormone and in the birth of the field of endocrinol. The secretin receptor has become prototypic of this receptor family, binding a moderately long linear peptide with a diffuse pharmacophoric domain. Here, we provide a detailed account of the contributions of photoaffinity labeling to establish the mol. basis of natural ligand binding to this receptor, as well as to provide insights into possible mechanisms for receptor activation and initiation of signaling. Each of the themes discussed are also relevant to other members of this physiol. and pharmacol. important receptor family.
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  17. 17
    Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001) Expanding the genetic code of Escherichia coli. Science 292, 498– 500,  DOI: 10.1126/science.1060077
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    17
    Expanding the genetic code of Escherichia coli
    Wang, Lei; Brock, Ansgar; Herberich, Brad; Schultz, Peter G.
    Science (Washington, DC, United States) (2001), 292 (5516), 498-500CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A unique tRNA/aminoacyl-tRNA synthetase pair has been generated that expands the no. of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as detd. by anal. of dihydrofolate reductase contg. the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chem., and phys. properties not found in the common 20 amino acids.
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  18. 18
    Grunbeck, A. and Sakmar, T. P. (2013) Probing G protein-coupled receptor-ligand interactions with targeted photoactivatable cross-linkers. Biochemistry 52, 8625– 8632,  DOI: 10.1021/bi401300y
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    18
    Probing G Protein-Coupled Receptor-Ligand Interactions with Targeted Photoactivatable Cross-Linkers
    Grunbeck, Amy; Sakmar, Thomas P.
    Biochemistry (2013), 52 (48), 8625-8632CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    A review. It has been 50 years since F.H.Westheimer and colleagues reported the first use of a photoactivatable crosslinking reagent to study the active site of chymotrypsin. In studies of seven transmembrane helical receptors, also known as G protein-coupled receptors (GPCRs), recent simultaneous advances in structural biol., mol. dynamics simulations, and amber codon suppression methods have allowed the development of a targeted photo-crosslinking strategy to probe receptor-ligand interactions in cell membranes. We review here recent advances in targeted photo-crosslinking of GPCR-ligand complexes in the context of extensive earlier work that primarily relied upon the use of ligand analogs with photoactivatable constituents.
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  19. 19
    Valentin-Hansen, L., Park, M., Huber, T., Grunbeck, A., Naganathan, S., Schwartz, T. W., and Sakmar, T. P. (2014) Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid. J. Biol. Chem. 289, 18045– 18054,  DOI: 10.1074/jbc.M113.527085
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    19
    Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid
    Valentin-Hansen, Louise; Park, Minyoung; Huber, Thomas; Grunbeck, Amy; Naganathan, Saranga; Schwartz, Thue W.; Sakmar, Thomas P.
    Journal of Biological Chemistry (2014), 289 (26), 18045-18054CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Substance P (SP) is a neuropeptide that mediates numerous physiol. responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogs suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, the authors applied a novel receptor-based targeted photocross-linking approach. The authors used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-l-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11-21) and 23 positions in the ECLII (residues 170(C-10)-193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII crosslinked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format.
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  20. 20
    Park, M., Sivertsen, B. B., Els-Heindl, S., Huber, T., Holst, B., Beck-Sickinger, A. G., Schwartz, T. W., and Sakmar, T. P. (2015) Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics. Chem. Biol. 22, 1431– 1436,  DOI: 10.1016/j.chembiol.2015.09.014
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    20
    Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics
    Park, Minyoung; Sivertsen, Bjoern B.; Els-Heindl, Sylvia; Huber, Thomas; Holst, Birgitte; Beck-Sickinger, Annette G.; Schwartz, Thue W.; Sakmar, Thomas P.
    Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (11), 1431-1436CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems.
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  21. 21
    Coin, I., Katritch, V., Sun, T., Xiang, Z., Siu, F. Y., Beyermann, M., Stevens, R. C., and Wang, L. (2013) Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR. Cell 155, 1258– 1269,  DOI: 10.1016/j.cell.2013.11.008
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    21
    Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR
    Coin, Irene; Katritch, Vsevolod; Sun, Tingting; Xiang, Zheng; Siu, Fai Yiu; Beyermann, Michael; Stevens, Raymond C.; Wang, Lei
    Cell (Cambridge, MA, United States) (2013), 155 (6), 1258-1269CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Mol. determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. The authors have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochem. and new click-chem. probes into the intact receptor expressed in the native membrane of live cells, 44 intermol. spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield mol. insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.
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  22. 22
    Seidel, L., Zarzycka, B., Zaidi, S. A., Katritch, V., and Coin, I. (2017) Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells. eLife 6, e27711,  DOI: 10.7554/eLife.27711
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    22
    Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells
    Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A.; Katritch, Vsevolod; Coin, Irene
    eLife (2017), 6 (), e27711/1-e27711/25CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by mol. dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helixes VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors.
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    Coin, I., Perrin, M. H., Vale, W. W., and Wang, L. (2011) Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison, Angew Chem Int Ed Engl. Angew. Chem., Int. Ed. 50, 8077– 8081,  DOI: 10.1002/anie.201102646
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    Koole, C., Reynolds, C. A., Mobarec, J. C., Hick, C., Sexton, P. M., and Sakmar, T. P. (2017) Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R). J. Biol. Chem. 292, 7131– 7144,  DOI: 10.1074/jbc.M117.779496
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    24
    Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)
    Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.
    Journal of Biological Chemistry (2017), 292 (17), 7131-7144CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biol. activity of the GLP-1R with the mol. structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-L-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to det. the proximity of the azido group in the mutant receptor with the peptide exendin-4. Crosslinking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9-39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a mol. model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with addnl. exptl. constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state.
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    Hay, D. L., Harris, P. W., Kowalczyk, R., Brimble, M. A., Rathbone, D. L., Barwell, J., Conner, A. C., and Poyner, D. R. (2014) Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation. Br. J. Pharmacol. 171, 415– 426,  DOI: 10.1111/bph.12464
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    25
    Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation
    Hay, Debbie L.; Harris, Paul W. R.; Kowalczyk, Renata; Brimble, Margaret A.; Rathbone, Dan L.; Barwell, James; Conner, Alex C.; Poyner, David R.
    British Journal of Pharmacology (2014), 171 (2), 415-426CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
    Background and Purpose : The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, esp. the disulfide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examd. and so the mol. determinants of agonism are unknown. This study has examd. the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Exptl. Approach : CGRP derivs. were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP prodn. in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key Results : Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for prodn. of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approx. 10-fold redns. in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and Implications : Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.
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    Wöhr, T. and Mutter, M. (1995) Pseudo-prolines in peptide synthesis: Direct insertion of serine and threonine derived oxazolidines in dipeptides. Tetrahedron Lett. 36, 3847– 3848,  DOI: 10.1016/0040-4039(95)00667-2
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    There is no corresponding record for this reference.
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    Fischer, R., Mader, O., Jung, G., and Brock, R. (2003) Extending the Applicability of Carboxyfluorescein in Solid-Phase Synthesis. Bioconjugate Chem. 14, 653– 660,  DOI: 10.1021/bc025658b
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    Extending the Applicability of Carboxyfluorescein in Solid-Phase Synthesis
    Fischer, Rainer; Mader, Oliver; Jung, Guenther; Brock, Roland
    Bioconjugate Chemistry (2003), 14 (3), 653-660CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in-situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing post-synthesis workup. For the cost-efficient labeling of large compd. collections, coupling protocols were developed reducing the amt. of coupling reagent and fluorophore. To enable further chem. derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanal. applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compd. collections with uniform labeling yields.
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    Harris, P. W. R., Lee, D. J., and Brimble, M. A. (2012) A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography,. J. Pept. Sci. 18, 549– 555,  DOI: 10.1002/psc.2432
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    A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography
    Harris, Paul W. R.; Lee, Dong Jun; Brimble, Margaret A.
    Journal of Peptide Science (2012), 18 (9), 549-555CODEN: JPSIEI; ISSN:1075-2617. (John Wiley & Sons Ltd.)
    Unquestionably, the purifn. of polypeptides by chromatog. methods is a considerable bottleneck in their prepn. Peptides synthesized by solid phase synthesis typically contain chromatog. similar impurities that complicate purifn. by reversed phase HPLC techniques. The authors report on the application of a slow gradient HPLC protocol that allows, in a single chromatog. step, the purifn. of hundreds of milligrams of material. This technique was applied to an extensive collection of synthetic polypeptides, some incorporating nonproteinogenic functionality. In all cases examd., the peptides were not only obtained in high purity, but were also recovered in multi-milligram amts. Copyright © 2012 European Peptide Society and John Wiley and Sons, Ltd.
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    Ye, S., Kohrer, C., Huber, T., Kazmi, M., Sachdev, P., Yan, E. C., Bhagat, A., RajBhandary, U. L., and Sakmar, T. P. (2008) Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J. Biol. Chem. 283, 1525– 1533,  DOI: 10.1074/jbc.M707355200
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    Garelja, M. L., Walker, C. A., Siow, A., Yang, S. H., Harris, P. W. R., Brimble, M. A., Watkins, H. A., Gingell, J. J., and Hay, D. L. (2018) Receptor Activity Modifying Proteins Have Limited Effects on the Class B G Protein-Coupled Receptor Calcitonin Receptor-Like Receptor Stalk. Biochemistry 57, 1410– 1422,  DOI: 10.1021/acs.biochem.7b01180
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    30
    Receptor Activity Modifying Proteins Have Limited Effects on the Class B G Protein-Coupled Receptor Calcitonin Receptor-Like Receptor Stalk
    Garelja, Michael L.; Walker, Christina A.; Siow, Andrew; Yang, Sung H.; Harris, Paul W. R.; Brimble, Margaret A.; Watkins, Harriet A.; Gingell, Joseph J.; Hay, Debbie L.
    Biochemistry (2018), 57 (8), 1410-1422CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The calcitonin receptor-like receptor (CLR) is a class B G protein-coupled receptor (GPCR) that forms the basis of three pharmacol. distinct receptors, the calcitonin gene-related peptide (CGRP) receptor, and two adrenomedullin (AM) receptors. These three receptors are created by CLR interacting with three receptor activity-modifying proteins (RAMPs). Class B GPCRs have an N-terminal extracellular domain (ECD) and transmembrane bundle that are both important for binding endogenous ligands. These two domains are joined together by a stretch of amino acids that is referred to as the "stalk". Studies of other class B GPCRs suggest that the stalk may act as hinge, allowing the ECD to adopt multiple conformations. It is unclear what the role of the stalk is within CLR and whether RAMPs can influence its function. Therefore, this study investigated the role of this region using an alanine scan. Effects of mutations were measured with all three RAMPs through cell surface expression, cAMP prodn. and, in select cases, radioligand binding and total cell expression assays. Most mutants did not affect expression or cAMP signaling. CLR C127A, N140A, F142A, and L144A impaired cell surface expression with all three RAMPs. T125A decreased the potency of all peptides at all receptors. N128A, V135A, and L139A showed ligand-dependent effects. While the stalk appears to play a role in CLR function, the effect of RAMPs on this region seems limited, in contrast to their effects on the structure of CLR in other receptor regions.
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    Qi, T., Christopoulos, G., Bailey, R. J., Christopoulos, A., Sexton, P. M., and Hay, D. L. (2008) Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function. Mol. Pharmacol. 74, 1059– 1071,  DOI: 10.1124/mol.108.047142
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    31
    Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function
    Qi, Tao; Christopoulos, George; Bailey, Richard J.; Christopoulos, Arthur; Sexton, Patrick M.; Hay, Debbie L.
    Molecular Pharmacology (2008), 74 (4), 1059-1071CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)
    Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, resp.). How RAMPs change G protein-coupled receptor pharmacol. is not fully understood. The authors exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacol. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. The authors hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, the authors introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-neg. form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP prodn. and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was crit. for detg. AM potency and affinity, and Phe 93 in RAMP1 was an important contributor to αCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacol.
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    Sali, A. and Blundell, T. L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779– 815,  DOI: 10.1006/jmbi.1993.1626
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    Comparative protein modeling by satisfaction of spatial restraints
    Sali, Andrej; Blundell, Tom L.
    Journal of Molecular Biology (1993), 234 (3), 779-815CODEN: JMOBAK; ISSN:0022-2836.
    The authors describe a comparative protein modeling method designed to find the most probable structure for a sequence given its alignment with related structures. The three-dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability d. functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equiv. residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain Cα-Cα distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the mol. pdf such that the model violates the input restraints as little as possible. The mol. pdf is derived as a combination of pdfs restraining individual spatial features of the whole mol. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non-hydrogen atoms. The method is automated and is illustrated by the modeling of trypsin from two other serine proteinases.
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    Barth, P., Schonbrun, J., and Baker, D. (2007) Toward high-resolution prediction and design of transmembrane helical protein structures. Proc. Natl. Acad. Sci. U. S. A. 104, 15682– 15687,  DOI: 10.1073/pnas.0702515104
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    Toward high-resolution prediction and design of transmembrane helical protein structures
    Barth, P.; Schonbrun, J.; Bakert, D.
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (40), 15682-15687CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The prediction and design at the at. level of membrane protein structures and interactions is a crit. but unsolved challenge. To address this problem, we have developed an all-atom phys. model that describes intraprotein and protein-solvent interactions in the membrane environment. We evaluated the ability of the model to recapitulate the energetics and structural specificities of polytopic membrane proteins by using a battery of in silico prediction and design tests. First, in side-chain packing and design tests, the model successfully predicts the side-chain conformations at 73% of nonexposed positions and the native amino acid identities at 34% of positions in naturally occurring membrane proteins. Second, the model predicts significant energy gaps between native and nonnative structures of transmembrane helical interfaces and polytopic membrane proteins. Third, distortions in trans-membrane helixes are successfully recapitulated in docking expts. by using fragments of ideal helixes judiciously defined around helical kinks. Finally, de novo structure prediction reaches near-at. accuracy (< 2.5 A) for several small membrane protein domains (< 150 residues). The success of the model highlights the crit. role of van der Waals and hydrogen-bonding interactions in the stability and structural specificity of membrane protein structures and sets the stage for the high-resoln. prediction and design of complex membrane protein architectures.
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    Watkins, H. A., Rathbone, D. L., Barwell, J., Hay, D. L., and Poyner, D. R. (2013) Structure-activity relationships for alpha-calcitonin gene-related peptide. Br. J. Pharmacol. 170, 1308– 1322,  DOI: 10.1111/bph.12072
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    Structure-activity relationships for α-calcitonin gene-related peptide
    Watkins, Harriet A.; Rathbone, Dan L.; Barwell, James; Hay, Debbie L.; Poyner, David R.
    British Journal of Pharmacology (2013), 170 (7), 1308-1322CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
    A review. Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulfide-bonded ring which is essential for biol. activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger no. contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix.
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    Ulmschneider, J. P. and Ulmschneider, M. B. (2009) Sampling efficiency in explicit and implicit membrane environments studied by peptide folding simulations. Proteins: Struct., Funct., Genet. 75, 586– 597,  DOI: 10.1002/prot.22270
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    Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD - Visual Molecular Dynamics. J. Mol. Graphics 14, 33– 38,  DOI: 10.1016/0263-7855(96)00018-5
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    VDM: visual molecular dynamics
    Humphrey, William; Dalke, Andrew; Schulten, Klaus
    Journal of Molecular Graphics (1996), 14 (1), 33-8, plates, 27-28CODEN: JMGRDV; ISSN:0263-7855. (Elsevier)
    VMD is a mol. graphics program designed for the display and anal. of mol. assemblies, in particular, biopolymers such as proteins and nucleic acids. VMD can simultaneously display any no. of structures using a wide variety of rendering styles and coloring methods. Mols. are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resoln. raster images of displayed mols. may be produced by generating input scripts for use by a no. of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate mol. dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biol., which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs, VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
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    Howitt, S. G., Kilk, K., Wang, Y., Smith, D. M., Langel, U., and Poyner, D. R. (2003) The role of the 8–18 helix of CGRP8–37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions,. Br. J. Pharmacol. 138, 325– 332,  DOI: 10.1038/sj.bjp.0705040
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    The role of the 8-18 helix of CGRP8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions
    Howitt, Stephen G.; Kilk, Kalle; Wang, Yang; Smith, David M.; Langel, Ulo; Poyner, David R.
    British Journal of Pharmacology (2003), 138 (2), 325-332CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
    The role of individual residues in the 8-18 helix of CGRP8-37 in promoting high-affinity binding to CGRP1 receptors expressed on rat L6 and human SK-N-MC cells has been examd. The relative potencies of various derivs. were estd. from their ability to inhibit the human αCGRP-mediated increase in cAMP prodn. and the binding of [125I]-human αCGRP. Arg11 and Arg18 were replaced by serines to give [Ser11,18]CGRP8-37. These bound with pKi values < 6 to SK-N-MC cells and had apparent pA2 values of 5.81±0.04 and 5.31±0.11 on SK-N-MC and L6 cells. CGRP8-37 had a pKi of 8.22 on SK-N-MC cells and pKb values on the above cell lines of 8.95±0.04 and 8.76±0.04. The arginines were replaced with glutamic acid residues. [Glu11]CGRP8-37 had a pKb of 7.14±0.14 on SK-N-MC cells (pKi = 7.05±0.05) and 6.99±0.08 on L6 cells. [Glu18]CGRP8-37 had a pKb of 7.10±0.0.08 on SK-N-MC cells (pKi = 6.91±0.23) and 7.12±0.09 on L6 cells. Leu12, Leu15 and Leu16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA2 values of [bpa12]-, [bpa15]- and [bpa16]CGRP8-37 were resp. 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and < 6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n = 3). It is concluded that the Arg11 and Arg18 are involved in specific electrostatic interactions with other residues, either on the CGRP1 receptors or elsewhere on CGRP8-37. Leu16 is in a conformationally restricted site when CGRP8-37 binds to CGRP1 receptors, unlike Leu12 and Leu15.
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    Watkins, H. A., Chakravarthy, M., Abhayawardana, R. S., Gingell, J. J., Garelja, M., Pardamwar, M., McElhinney, J. M., Lathbridge, A., Constantine, A., Harris, P. W., Yuen, T. Y., Brimble, M. A., Barwell, J., Poyner, D. R., Woolley, M. J., Conner, A. C., Pioszak, A. A., Reynolds, C. A., and Hay, D. L. (2016) Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties. J. Biol. Chem. 291, 11657– 11675,  DOI: 10.1074/jbc.M115.688218
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    Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties
    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.; Hay, Debbie L.
    Journal of Biological Chemistry (2016), 291 (22), 11657-11675CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), resp. These different CLR-RAMP interactions yield discrete receptor pharmacol. and physiol. effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the mol. details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and detd. the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent mol. modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability anal. indicated unique features that could be used to develop selective small mol. ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.
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    Balo, A. R., Wang, M., and Ernst, O. P. (2017) Accessible virtual reality of biomolecular structural models using the Autodesk Molecule Viewer. Nat. Methods 14, 1122– 1123,  DOI: 10.1038/nmeth.4506
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    Accessible virtual reality of biomolecular structural models using the Autodesk Molecule Viewer
    Balo, Aidin R.; Wang, Merry; Ernst, Oliver P.
    Nature Methods (2017), 14 (12), 1122-1123CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    There is no expanded citation for this reference.
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    Barwell, J., Conner, A., and Poyner, D. R. (2011) Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim. Biophys. Acta, Mol. Cell Res. 1813, 1906– 1916,  DOI: 10.1016/j.bbamcr.2011.06.005
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    Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function
    Barwell, James; Conner, Alex; Poyner, David R.
    Biochimica et Biophysica Acta, Molecular Cell Research (2011), 1813 (10), 1906-1916CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-assocd. transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which assocs. with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP prodn. and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP prodn.; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a no. of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP prodn. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.
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    Barwell, J., Miller, P. S., Donnelly, D., and Poyner, D. R. (2010) Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor. Peptides 31, 170– 176,  DOI: 10.1016/j.peptides.2009.10.021
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    Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor
    Barwell, James; Miller, Philip S.; Donnelly, Dan; Poyner, David R.
    Peptides (New York, NY, United States) (2010), 31 (1), 170-176CODEN: PPTDD5; ISSN:0196-9781. (Elsevier)
    The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examd. by an alanine scan and the results were interpreted with the help of a mol. model. The potency of CGRP at stimulating cAMP prodn. was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a sep. cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can assoc. with RAMPs.
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    Hoang, H. N., Song, K., Hill, T. A., Derksen, D. R., Edmonds, D. J., Kok, W. M., Limberakis, C., Liras, S., Loria, P. M., Mascitti, V., Mathiowetz, A. M., Mitchell, J. M., Piotrowski, D. W., Price, D. A., Stanton, R. V., Suen, J. Y., Withka, J. M., Griffith, D. A., and Fairlie, D. P. (2015) Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists. J. Med. Chem. 58, 4080– 4085,  DOI: 10.1021/acs.jmedchem.5b00166
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    Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists
    Hoang, Huy N.; Song, Kun; Hill, Timothy A.; Derksen, David R.; Edmonds, David J.; Kok, W. Mei; Limberakis, Chris; Liras, Spiros; Loria, Paula M.; Mascitti, Vincent; Mathiowetz, Alan M.; Mitchell, Justin M.; Piotrowski, David W.; Price, David A.; Stanton, Robert V.; Suen, Jacky Y.; Withka, Jane M.; Griffith, David A.; Fairlie, David P.
    Journal of Medicinal Chemistry (2015), 58 (9), 4080-4085CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Cyclic constraints are incorporated into an 11-residue analog of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concns. in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small mol. agonists of the GLP-1 receptor to treat type 2 diabetes.
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    Dal Maso, E., Zhu, Y., Pham, V., Reynolds, C. A., Deganutti, G., Hick, C. A., Yang, D., Christopoulos, A., Hay, D. L., Wang, M. W., Sexton, P. M., Furness, S. G. B., and Wootten, D. (2018) Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy. Biochem. Pharmacol. 150, 214– 244,  DOI: 10.1016/j.bcp.2018.02.005
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    Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy
    Dal Maso, Emma; Zhu, Yue; Pham, Vi; Reynolds, Christopher A.; Deganutti, Giuseppe; Hick, Caroline A.; Yang, Dehua; Christopoulos, Arthur; Hay, Debbie L.; Wang, Ming-Wei; Sexton, Patrick M.; Furness, Sebastian G. B.; Wootten, Denise
    Biochemical Pharmacology (Amsterdam, Netherlands) (2018), 150 (), 214-244CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
    Class B peptide hormone GPCRs are targets for the treatment of major chronic disease. Peptide ligands of these receptors display biased agonism and this may provide future therapeutic advantage. Recent active structures of the calcitonin (CT) and glucagon-like peptide-1 (GLP-1) receptors reveal distinct engagement of peptides with extracellular loops (ECLs) 2 and 3, and mutagenesis of the GLP-1R has implicated these loops in dynamics of receptor activation. In the current study, we have mutated ECLs 2 and 3 of the human CT receptor (CTR), to interrogate receptor expression, peptide affinity and efficacy. Integration of these data with insights from the CTR and GLP-1R active structures, revealed marked diversity in mechanisms of peptide engagement and receptor activation between the CTR and GLP-1R. While the CTR ECL2 played a key role in conformational propagation linked to Gs/cAMP signalling this was mechanistically distinct from that of GLP-1R ECL2. Moreover, ECL3 was a hot-spot for distinct ligand- and pathway-specific effects, and this has implications for the future design of biased agonists of class B GPCRs.
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    Wootten, D., Reynolds, C. A., Smith, K. J., Mobarec, J. C., Koole, C., Savage, E. E., Pabreja, K., Simms, J., Sridhar, R., Furness, S. G., Liu, M., Thompson, P. E., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2016) The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism,. Cell 165, 1632– 1643,  DOI: 10.1016/j.cell.2016.05.023
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    Gkountelias, K., Tselios, T., Venihaki, M., Deraos, G., Lazaridis, I., Rassouli, O., Gravanis, A., and Liapakis, G. (2009) Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding. Mol. Pharmacol. 75, 793– 800,  DOI: 10.1124/mol.108.052423
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    Hollenstein, K., Kean, J., Bortolato, A., Cheng, R. K., Dore, A. S., Jazayeri, A., Cooke, R. M., Weir, M., and Marshall, F. H. (2013) Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438– 443,  DOI: 10.1038/nature12357
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    Structure of class B GPCR corticotropin-releasing factor receptor 1
    Hollenstein, Kaspar; Kean, James; Bortolato, Andrea; Cheng, Robert K. Y.; Dore, Andrew S.; Jazayeri, Ali; Cooke, Robert M.; Weir, Malcolm; Marshall, Fiona H.
    Nature (London, United Kingdom) (2013), 499 (7459), 438-443CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Structural anal. of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 (CRF1R) is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-mol. antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-mol. drugs for diseases of brain and metab.
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  • Abstract

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    Figure 1

    Figure 1. cAMP production on HEK293T cells transfected with CLR and RAMP1 and exposed to CGRP or 15-Fluo CGRP for 15 min. Values are means ± the standard error of the mean (SEM); n = 3 independent experiments.

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    Figure 2

    Figure 2. Fluorescence of solubilized HEK293T cell membranes from cells transfected with RAMP1 and CLR containing azF substitutions at the indicated residues. Membranes were exposed to 15-Fluo CGRP in the presence or absence of UV light. D94, H114, W121, and N128 are all within 5 Å of bound CGRP; (10) other residues are 7–10 Å away. Values are means ± SEM; n = 3. *P < 0.05, and **P < 0.01; values of total labeling that are significantly different from the nonspecific values in the paired control samples, i.e., in the absence of UV light.

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    Figure 3

    Figure 3. Fluorescence of solubilized HEK293T cell membranes from cells transfected with RAMP1 and CLR containing azF substitutions at the indicated residues. Membranes were exposed to 15-Fluo CGRP in the presence or absence of UV light. The values are the specific labeling, following subtraction of the nonspecific from total bound fluorescence. Values are means ± SEM; n = 3–6. *P < 0.05; values for which total labeling is significantly different from the nonspecific values in the paired control samples, i.e., in the absence of UV light. Nonspecific labeling ranged from 2247 to 5759 AU.

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    Figure 4

    Figure 4. Tyrosine scan of ECL2. Potency of human α-CGRP at stimulating cAMP production in HEK293T cells transfected with CLR (WT or mutants) and RAMP1. Values are means ± SEM; n = 3.

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    Figure 5

    Figure 5. Fluorescence of solubilized HEK293T cell membranes from cells transfected with RAMP1 and CLR containing azF substitutions at the indicated residues. Membranes were exposed to 15-Fluo CGRP in the presence or absence of UV light. Values are means ± SEM; n = 3. *P < 0.05; values of total labeling significantly different from the nonspecific values in the paired control samples, i.e., in the absence of UV light.

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    Figure 6

    Figure 6. Molecular model of the CLR (brown ribbon)/RAMP1 (blue ribbon; TM region omitted for the sake of clarity) complex with bound CGRP (pink ribbon). (A) Detail of predicted interactions of CGRP with I284 and L291. (B) Predicted position of bound CGRP with residues on the periphery of the binding site. CLR residues shown in space-filling mode; CGRP residues colored green. The models can be viewed online using Molecule Viewer (39) at https://bit.ly/2FnQAHp (recommended browsers are Google Chrome in incognito mode or Firefox). (C) Full length structure of the CLR complex. The positions of TM5 and helix 8 are highlighted to aid orientation.

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    Figure 7

    Figure 7. Comparison of the ability of [Leu12Ala]- and [Leu16Ala]-human α-CGRP (A) to stimulate cAMP in HEK293S cells transfected with HA-CLR and myc-RAMP1 or (B) to displace [125I]CGRP at the CGRP receptor. Values are means ± SEM; n = 3 or 4.

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  • References

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    This article references 46 other publications.

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      The calcitonin/CGRP family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two GPCRs, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerization of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM1 and AM2 receptors (CLR/RAMP2 or RAMP3) and the AMY1, AMY2 and AMY3 receptors (CTR/RAMPs1-3 complexes, resp.). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors, and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT(a) and CT(b)). Furthermore, the AMY1(a) receptor is activated equally well by both amylin and CGRP, and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example, many agents targeting the CGRP system are in clin. trials, and pramlintide, an amylin analog, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacol. of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clin. Pharmacol. and colleagues.
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      Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resoln. limitations, the specific mol. interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding interdomain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resoln. crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analog and partial agonist, NNC1702. This structure provides mol. details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual binding-site trigger model for GCGR activation - which requires conformational changes of the stalk, first extracellular loop and TMD - that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.
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      Nature (London, United Kingdom) (2018), 555 (7694), 121-125CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gαs heterotrimer, detd. at a global resoln. of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure. At the intracellular face, there was a six-degree difference in the angle of the Gαs-α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addn., the structures differed in the rate and extent of conformational reorganization of the Gαs protein. Our structure provides insights into the mol. basis of biased agonism.
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      9
      Phase-plate cryo-EM structure of a class B GPCR-G-protein complex
      Liang, Yi-Lynn; Khoshouei, Maryam; Radjainia, Mazdak; Zhang, Yan; Glukhova, Alisa; Tarrasch, Jeffrey; Thal, David M.; Furness, Sebastian G. B.; Christopoulos, George; Coudrat, Thomas; Danev, Radostin; Baumeister, Wolfgang; Miller, Laurence J.; Christopoulos, Arthur; Kobilka, Brian K.; Wootten, Denise; Skiniotis, Georgios; Sexton, Patrick M.
      Nature (London, United Kingdom) (2017), 546 (7656), 118-123CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Class B G-protein-coupled receptors (GPCRs) are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes, and obesity. Here, we report the structure of a full-length class B GPCR receptor, the calcitonin receptor, in complex with the peptide ligand, calcitonin, and a heterotrimeric Gαsβγ protein detd. by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist, calcitonin, engaged the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helixes 6 and 7. This conformation was accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also obsd. was an extended intracellular helix 8 that contributed to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. Thus, this structure provides a new framework for understanding GPCR receptor function.
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    10. 10
      Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor. Mol. Cell 58, 1040– 1052,  DOI: 10.1016/j.molcel.2015.04.018
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      10
      Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor
      Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.
      Molecular Cell (2015), 58 (6), 1040-1052CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Assocn. of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolns., resp. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacol. data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.
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    11. 11
      Woolley, M. J., Reynolds, C. A., Simms, J., Walker, C. S., Mobarec, J. C., Garelja, M. L., Conner, A. C., Poyner, D. R., and Hay, D. L. (2017) Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs. Biochem. Pharmacol. 142, 96– 110,  DOI: 10.1016/j.bcp.2017.07.005
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      11
      Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs
      Woolley, Michael J.; Reynolds, Christopher A.; Simms, John; Walker, Christopher S.; Mobarec, Juan Carlos; Garelja, Michael L.; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.
      Biochemical Pharmacology (Amsterdam, Netherlands) (2017), 142 (), 96-110CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
      Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP prodn., modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting mol. dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The mol. dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.
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    12. 12
      Woolley, M. J., Simms, J., Mobarec, J. C., Reynolds, C. A., Poyner, D. R., and Conner, A. C. (2017) Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach. Mol. Cell. Endocrinol. 454, 39– 49,  DOI: 10.1016/j.mce.2017.05.034
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      12
      Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach
      Woolley, Michael J.; Simms, John; Mobarec, Juan Carlos; Reynolds, Christopher A.; Poyner, David R.; Conner, Alex C.
      Molecular and Cellular Endocrinology (2017), 454 (), 39-49CODEN: MCEND6; ISSN:0303-7207. (Elsevier Ireland Ltd.)
      The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution anal. method and mol. modeling to the functionally important residues of ECL2 to reveal key mol. interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest redn. in signalling following alanine substitution anal. and comprise a group of basic, acidic and arom. residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochem. requirements for neighboring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.
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    13. 13
      Woolley, M. J., Simms, J., Uddin, S., Poyner, D. R., and Conner, A. C. (2017) Relative Antagonism of Mutants of the CGRP Receptor Extracellular Loop 2 Domain (ECL2) Using a Truncated Competitive Antagonist (CGRP8–37): Evidence for the Dual Involvement of ECL2 in the Two-Domain Binding Model. Biochemistry 56, 3877– 3880,  DOI: 10.1021/acs.biochem.7b00077
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      There is no corresponding record for this reference.
    14. 14
      Woolley, M. J., Watkins, H. A., Taddese, B., Karakullukcu, Z. G., Barwell, J., Smith, K. J., Hay, D. L., Poyner, D. R., Reynolds, C. A., and Conner, A. C. (2013) The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach,. J. R. Soc., Interface 10, 20130589,  DOI: 10.1098/rsif.2013.0589
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      14
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach
      Woolley, Michael J.; Watkins, Harriet A.; Taddese, Bruck; Karakullukcu, Z. Gamze; Barwell, James; Smith, Kevin J.; Hay, Debbie L.; Poyner, David R.; Reynolds, Christopher A.; Conner, Alex C.
      Journal of the Royal Society, Interface (2013), 10 (88), 20130589/1-20130589/11CODEN: JRSICU; ISSN:1742-5689. (Royal Society)
      The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modeling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.
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    15. 15
      Vohra, S., Taddese, B., Conner, A. C., Poyner, D. R., Hay, D. L., Barwell, J., Reeves, P. J., Upton, G. J., and Reynolds, C. A. (2013) Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies, Journal of the Royal Society. J. R. Soc., Interface 10, 20120846,  DOI: 10.1098/rsif.2012.0846
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      15
      Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies
      Vohra, Shabana; Taddese, Bruck; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.; Barwell, James; Reeves, Philip J.; Upton, Graham J. G.; Reynolds, Christopher A.
      Journal of the Royal Society, Interface (2013), 10 (79), 20120846/1-20120846/13CODEN: JRSICU; ISSN:1742-5689. (Royal Society)
      Modeling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homol. The plant GPCR, GCR1, has homol. to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating max. lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this anal. to generate active and inactive homol. models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the anal. of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to assoc. the key mol. features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg2.39, His2.43 and Glu3.46, which makes a polar lock with T6.37. These alignments and models provide useful tools for understanding class B GPCR function.
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    16. 16
      Dong, M. and Miller, L. J. (2006) Use of photoaffinity labeling to understand the molecular basis of ligand binding to the secretin receptor. Ann. N. Y. Acad. Sci. 1070, 248– 264,  DOI: 10.1196/annals.1317.023
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      16
      Use of photoaffinity labeling to understand the molecular basis of ligand binding to the secretin receptor
      Dong, Maoqing; Miller, Laurence J.
      Annals of the New York Academy of Sciences (2006), 1070 (VIP, PACAP, and Related Peptides), 248-264CODEN: ANYAA9; ISSN:0077-8923. (Blackwell Publishing, Inc.)
      A review. The secretin receptor was the first member of the Class B family of G protein-coupled receptors that was identified in 1991, 89 years after secretin action was first recognized. That report resulted in the introduction of the term hormone and in the birth of the field of endocrinol. The secretin receptor has become prototypic of this receptor family, binding a moderately long linear peptide with a diffuse pharmacophoric domain. Here, we provide a detailed account of the contributions of photoaffinity labeling to establish the mol. basis of natural ligand binding to this receptor, as well as to provide insights into possible mechanisms for receptor activation and initiation of signaling. Each of the themes discussed are also relevant to other members of this physiol. and pharmacol. important receptor family.
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    17. 17
      Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001) Expanding the genetic code of Escherichia coli. Science 292, 498– 500,  DOI: 10.1126/science.1060077
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      17
      Expanding the genetic code of Escherichia coli
      Wang, Lei; Brock, Ansgar; Herberich, Brad; Schultz, Peter G.
      Science (Washington, DC, United States) (2001), 292 (5516), 498-500CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A unique tRNA/aminoacyl-tRNA synthetase pair has been generated that expands the no. of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as detd. by anal. of dihydrofolate reductase contg. the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chem., and phys. properties not found in the common 20 amino acids.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjtVenu78%253D&md5=2f01bbe1fb271afd34f43351f83129f6
    18. 18
      Grunbeck, A. and Sakmar, T. P. (2013) Probing G protein-coupled receptor-ligand interactions with targeted photoactivatable cross-linkers. Biochemistry 52, 8625– 8632,  DOI: 10.1021/bi401300y
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      18
      Probing G Protein-Coupled Receptor-Ligand Interactions with Targeted Photoactivatable Cross-Linkers
      Grunbeck, Amy; Sakmar, Thomas P.
      Biochemistry (2013), 52 (48), 8625-8632CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      A review. It has been 50 years since F.H.Westheimer and colleagues reported the first use of a photoactivatable crosslinking reagent to study the active site of chymotrypsin. In studies of seven transmembrane helical receptors, also known as G protein-coupled receptors (GPCRs), recent simultaneous advances in structural biol., mol. dynamics simulations, and amber codon suppression methods have allowed the development of a targeted photo-crosslinking strategy to probe receptor-ligand interactions in cell membranes. We review here recent advances in targeted photo-crosslinking of GPCR-ligand complexes in the context of extensive earlier work that primarily relied upon the use of ligand analogs with photoactivatable constituents.
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    19. 19
      Valentin-Hansen, L., Park, M., Huber, T., Grunbeck, A., Naganathan, S., Schwartz, T. W., and Sakmar, T. P. (2014) Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid. J. Biol. Chem. 289, 18045– 18054,  DOI: 10.1074/jbc.M113.527085
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      19
      Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid
      Valentin-Hansen, Louise; Park, Minyoung; Huber, Thomas; Grunbeck, Amy; Naganathan, Saranga; Schwartz, Thue W.; Sakmar, Thomas P.
      Journal of Biological Chemistry (2014), 289 (26), 18045-18054CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Substance P (SP) is a neuropeptide that mediates numerous physiol. responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogs suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, the authors applied a novel receptor-based targeted photocross-linking approach. The authors used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-l-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11-21) and 23 positions in the ECLII (residues 170(C-10)-193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII crosslinked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format.
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    20. 20
      Park, M., Sivertsen, B. B., Els-Heindl, S., Huber, T., Holst, B., Beck-Sickinger, A. G., Schwartz, T. W., and Sakmar, T. P. (2015) Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics. Chem. Biol. 22, 1431– 1436,  DOI: 10.1016/j.chembiol.2015.09.014
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      20
      Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics
      Park, Minyoung; Sivertsen, Bjoern B.; Els-Heindl, Sylvia; Huber, Thomas; Holst, Birgitte; Beck-Sickinger, Annette G.; Schwartz, Thue W.; Sakmar, Thomas P.
      Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (11), 1431-1436CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems.
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    21. 21
      Coin, I., Katritch, V., Sun, T., Xiang, Z., Siu, F. Y., Beyermann, M., Stevens, R. C., and Wang, L. (2013) Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR. Cell 155, 1258– 1269,  DOI: 10.1016/j.cell.2013.11.008
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      21
      Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR
      Coin, Irene; Katritch, Vsevolod; Sun, Tingting; Xiang, Zheng; Siu, Fai Yiu; Beyermann, Michael; Stevens, Raymond C.; Wang, Lei
      Cell (Cambridge, MA, United States) (2013), 155 (6), 1258-1269CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Mol. determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. The authors have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochem. and new click-chem. probes into the intact receptor expressed in the native membrane of live cells, 44 intermol. spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield mol. insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.
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    22. 22
      Seidel, L., Zarzycka, B., Zaidi, S. A., Katritch, V., and Coin, I. (2017) Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells. eLife 6, e27711,  DOI: 10.7554/eLife.27711
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      22
      Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells
      Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A.; Katritch, Vsevolod; Coin, Irene
      eLife (2017), 6 (), e27711/1-e27711/25CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by mol. dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helixes VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors.
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    23. 23
      Coin, I., Perrin, M. H., Vale, W. W., and Wang, L. (2011) Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison, Angew Chem Int Ed Engl. Angew. Chem., Int. Ed. 50, 8077– 8081,  DOI: 10.1002/anie.201102646
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      There is no corresponding record for this reference.
    24. 24
      Koole, C., Reynolds, C. A., Mobarec, J. C., Hick, C., Sexton, P. M., and Sakmar, T. P. (2017) Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R). J. Biol. Chem. 292, 7131– 7144,  DOI: 10.1074/jbc.M117.779496
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      24
      Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)
      Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.
      Journal of Biological Chemistry (2017), 292 (17), 7131-7144CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biol. activity of the GLP-1R with the mol. structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-L-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to det. the proximity of the azido group in the mutant receptor with the peptide exendin-4. Crosslinking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9-39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a mol. model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with addnl. exptl. constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state.
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    25. 25
      Hay, D. L., Harris, P. W., Kowalczyk, R., Brimble, M. A., Rathbone, D. L., Barwell, J., Conner, A. C., and Poyner, D. R. (2014) Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation. Br. J. Pharmacol. 171, 415– 426,  DOI: 10.1111/bph.12464
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      25
      Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation
      Hay, Debbie L.; Harris, Paul W. R.; Kowalczyk, Renata; Brimble, Margaret A.; Rathbone, Dan L.; Barwell, James; Conner, Alex C.; Poyner, David R.
      British Journal of Pharmacology (2014), 171 (2), 415-426CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
      Background and Purpose : The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, esp. the disulfide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examd. and so the mol. determinants of agonism are unknown. This study has examd. the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Exptl. Approach : CGRP derivs. were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP prodn. in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key Results : Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for prodn. of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approx. 10-fold redns. in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and Implications : Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.
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      Wöhr, T. and Mutter, M. (1995) Pseudo-prolines in peptide synthesis: Direct insertion of serine and threonine derived oxazolidines in dipeptides. Tetrahedron Lett. 36, 3847– 3848,  DOI: 10.1016/0040-4039(95)00667-2
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      Fischer, R., Mader, O., Jung, G., and Brock, R. (2003) Extending the Applicability of Carboxyfluorescein in Solid-Phase Synthesis. Bioconjugate Chem. 14, 653– 660,  DOI: 10.1021/bc025658b
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      Extending the Applicability of Carboxyfluorescein in Solid-Phase Synthesis
      Fischer, Rainer; Mader, Oliver; Jung, Guenther; Brock, Roland
      Bioconjugate Chemistry (2003), 14 (3), 653-660CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in-situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing post-synthesis workup. For the cost-efficient labeling of large compd. collections, coupling protocols were developed reducing the amt. of coupling reagent and fluorophore. To enable further chem. derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanal. applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compd. collections with uniform labeling yields.
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      Harris, P. W. R., Lee, D. J., and Brimble, M. A. (2012) A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography,. J. Pept. Sci. 18, 549– 555,  DOI: 10.1002/psc.2432
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      A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography
      Harris, Paul W. R.; Lee, Dong Jun; Brimble, Margaret A.
      Journal of Peptide Science (2012), 18 (9), 549-555CODEN: JPSIEI; ISSN:1075-2617. (John Wiley & Sons Ltd.)
      Unquestionably, the purifn. of polypeptides by chromatog. methods is a considerable bottleneck in their prepn. Peptides synthesized by solid phase synthesis typically contain chromatog. similar impurities that complicate purifn. by reversed phase HPLC techniques. The authors report on the application of a slow gradient HPLC protocol that allows, in a single chromatog. step, the purifn. of hundreds of milligrams of material. This technique was applied to an extensive collection of synthetic polypeptides, some incorporating nonproteinogenic functionality. In all cases examd., the peptides were not only obtained in high purity, but were also recovered in multi-milligram amts. Copyright © 2012 European Peptide Society and John Wiley and Sons, Ltd.
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      Ye, S., Kohrer, C., Huber, T., Kazmi, M., Sachdev, P., Yan, E. C., Bhagat, A., RajBhandary, U. L., and Sakmar, T. P. (2008) Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J. Biol. Chem. 283, 1525– 1533,  DOI: 10.1074/jbc.M707355200
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      Garelja, M. L., Walker, C. A., Siow, A., Yang, S. H., Harris, P. W. R., Brimble, M. A., Watkins, H. A., Gingell, J. J., and Hay, D. L. (2018) Receptor Activity Modifying Proteins Have Limited Effects on the Class B G Protein-Coupled Receptor Calcitonin Receptor-Like Receptor Stalk. Biochemistry 57, 1410– 1422,  DOI: 10.1021/acs.biochem.7b01180
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      Receptor Activity Modifying Proteins Have Limited Effects on the Class B G Protein-Coupled Receptor Calcitonin Receptor-Like Receptor Stalk
      Garelja, Michael L.; Walker, Christina A.; Siow, Andrew; Yang, Sung H.; Harris, Paul W. R.; Brimble, Margaret A.; Watkins, Harriet A.; Gingell, Joseph J.; Hay, Debbie L.
      Biochemistry (2018), 57 (8), 1410-1422CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The calcitonin receptor-like receptor (CLR) is a class B G protein-coupled receptor (GPCR) that forms the basis of three pharmacol. distinct receptors, the calcitonin gene-related peptide (CGRP) receptor, and two adrenomedullin (AM) receptors. These three receptors are created by CLR interacting with three receptor activity-modifying proteins (RAMPs). Class B GPCRs have an N-terminal extracellular domain (ECD) and transmembrane bundle that are both important for binding endogenous ligands. These two domains are joined together by a stretch of amino acids that is referred to as the "stalk". Studies of other class B GPCRs suggest that the stalk may act as hinge, allowing the ECD to adopt multiple conformations. It is unclear what the role of the stalk is within CLR and whether RAMPs can influence its function. Therefore, this study investigated the role of this region using an alanine scan. Effects of mutations were measured with all three RAMPs through cell surface expression, cAMP prodn. and, in select cases, radioligand binding and total cell expression assays. Most mutants did not affect expression or cAMP signaling. CLR C127A, N140A, F142A, and L144A impaired cell surface expression with all three RAMPs. T125A decreased the potency of all peptides at all receptors. N128A, V135A, and L139A showed ligand-dependent effects. While the stalk appears to play a role in CLR function, the effect of RAMPs on this region seems limited, in contrast to their effects on the structure of CLR in other receptor regions.
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      Qi, T., Christopoulos, G., Bailey, R. J., Christopoulos, A., Sexton, P. M., and Hay, D. L. (2008) Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function. Mol. Pharmacol. 74, 1059– 1071,  DOI: 10.1124/mol.108.047142
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      Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function
      Qi, Tao; Christopoulos, George; Bailey, Richard J.; Christopoulos, Arthur; Sexton, Patrick M.; Hay, Debbie L.
      Molecular Pharmacology (2008), 74 (4), 1059-1071CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)
      Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, resp.). How RAMPs change G protein-coupled receptor pharmacol. is not fully understood. The authors exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacol. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. The authors hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, the authors introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-neg. form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP prodn. and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was crit. for detg. AM potency and affinity, and Phe 93 in RAMP1 was an important contributor to αCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacol.
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    32. 32
      Sali, A. and Blundell, T. L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779– 815,  DOI: 10.1006/jmbi.1993.1626
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      Comparative protein modeling by satisfaction of spatial restraints
      Sali, Andrej; Blundell, Tom L.
      Journal of Molecular Biology (1993), 234 (3), 779-815CODEN: JMOBAK; ISSN:0022-2836.
      The authors describe a comparative protein modeling method designed to find the most probable structure for a sequence given its alignment with related structures. The three-dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability d. functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equiv. residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain Cα-Cα distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the mol. pdf such that the model violates the input restraints as little as possible. The mol. pdf is derived as a combination of pdfs restraining individual spatial features of the whole mol. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non-hydrogen atoms. The method is automated and is illustrated by the modeling of trypsin from two other serine proteinases.
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      Barth, P., Schonbrun, J., and Baker, D. (2007) Toward high-resolution prediction and design of transmembrane helical protein structures. Proc. Natl. Acad. Sci. U. S. A. 104, 15682– 15687,  DOI: 10.1073/pnas.0702515104
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      Toward high-resolution prediction and design of transmembrane helical protein structures
      Barth, P.; Schonbrun, J.; Bakert, D.
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (40), 15682-15687CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The prediction and design at the at. level of membrane protein structures and interactions is a crit. but unsolved challenge. To address this problem, we have developed an all-atom phys. model that describes intraprotein and protein-solvent interactions in the membrane environment. We evaluated the ability of the model to recapitulate the energetics and structural specificities of polytopic membrane proteins by using a battery of in silico prediction and design tests. First, in side-chain packing and design tests, the model successfully predicts the side-chain conformations at 73% of nonexposed positions and the native amino acid identities at 34% of positions in naturally occurring membrane proteins. Second, the model predicts significant energy gaps between native and nonnative structures of transmembrane helical interfaces and polytopic membrane proteins. Third, distortions in trans-membrane helixes are successfully recapitulated in docking expts. by using fragments of ideal helixes judiciously defined around helical kinks. Finally, de novo structure prediction reaches near-at. accuracy (< 2.5 A) for several small membrane protein domains (< 150 residues). The success of the model highlights the crit. role of van der Waals and hydrogen-bonding interactions in the stability and structural specificity of membrane protein structures and sets the stage for the high-resoln. prediction and design of complex membrane protein architectures.
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      Watkins, H. A., Rathbone, D. L., Barwell, J., Hay, D. L., and Poyner, D. R. (2013) Structure-activity relationships for alpha-calcitonin gene-related peptide. Br. J. Pharmacol. 170, 1308– 1322,  DOI: 10.1111/bph.12072
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      Structure-activity relationships for α-calcitonin gene-related peptide
      Watkins, Harriet A.; Rathbone, Dan L.; Barwell, James; Hay, Debbie L.; Poyner, David R.
      British Journal of Pharmacology (2013), 170 (7), 1308-1322CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
      A review. Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulfide-bonded ring which is essential for biol. activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger no. contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix.
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      Ulmschneider, J. P. and Ulmschneider, M. B. (2009) Sampling efficiency in explicit and implicit membrane environments studied by peptide folding simulations. Proteins: Struct., Funct., Genet. 75, 586– 597,  DOI: 10.1002/prot.22270
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      Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD - Visual Molecular Dynamics. J. Mol. Graphics 14, 33– 38,  DOI: 10.1016/0263-7855(96)00018-5
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      VDM: visual molecular dynamics
      Humphrey, William; Dalke, Andrew; Schulten, Klaus
      Journal of Molecular Graphics (1996), 14 (1), 33-8, plates, 27-28CODEN: JMGRDV; ISSN:0263-7855. (Elsevier)
      VMD is a mol. graphics program designed for the display and anal. of mol. assemblies, in particular, biopolymers such as proteins and nucleic acids. VMD can simultaneously display any no. of structures using a wide variety of rendering styles and coloring methods. Mols. are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resoln. raster images of displayed mols. may be produced by generating input scripts for use by a no. of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate mol. dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biol., which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs, VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
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      Howitt, S. G., Kilk, K., Wang, Y., Smith, D. M., Langel, U., and Poyner, D. R. (2003) The role of the 8–18 helix of CGRP8–37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions,. Br. J. Pharmacol. 138, 325– 332,  DOI: 10.1038/sj.bjp.0705040
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      The role of the 8-18 helix of CGRP8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions
      Howitt, Stephen G.; Kilk, Kalle; Wang, Yang; Smith, David M.; Langel, Ulo; Poyner, David R.
      British Journal of Pharmacology (2003), 138 (2), 325-332CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
      The role of individual residues in the 8-18 helix of CGRP8-37 in promoting high-affinity binding to CGRP1 receptors expressed on rat L6 and human SK-N-MC cells has been examd. The relative potencies of various derivs. were estd. from their ability to inhibit the human αCGRP-mediated increase in cAMP prodn. and the binding of [125I]-human αCGRP. Arg11 and Arg18 were replaced by serines to give [Ser11,18]CGRP8-37. These bound with pKi values < 6 to SK-N-MC cells and had apparent pA2 values of 5.81±0.04 and 5.31±0.11 on SK-N-MC and L6 cells. CGRP8-37 had a pKi of 8.22 on SK-N-MC cells and pKb values on the above cell lines of 8.95±0.04 and 8.76±0.04. The arginines were replaced with glutamic acid residues. [Glu11]CGRP8-37 had a pKb of 7.14±0.14 on SK-N-MC cells (pKi = 7.05±0.05) and 6.99±0.08 on L6 cells. [Glu18]CGRP8-37 had a pKb of 7.10±0.0.08 on SK-N-MC cells (pKi = 6.91±0.23) and 7.12±0.09 on L6 cells. Leu12, Leu15 and Leu16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA2 values of [bpa12]-, [bpa15]- and [bpa16]CGRP8-37 were resp. 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and < 6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n = 3). It is concluded that the Arg11 and Arg18 are involved in specific electrostatic interactions with other residues, either on the CGRP1 receptors or elsewhere on CGRP8-37. Leu16 is in a conformationally restricted site when CGRP8-37 binds to CGRP1 receptors, unlike Leu12 and Leu15.
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    38. 38
      Watkins, H. A., Chakravarthy, M., Abhayawardana, R. S., Gingell, J. J., Garelja, M., Pardamwar, M., McElhinney, J. M., Lathbridge, A., Constantine, A., Harris, P. W., Yuen, T. Y., Brimble, M. A., Barwell, J., Poyner, D. R., Woolley, M. J., Conner, A. C., Pioszak, A. A., Reynolds, C. A., and Hay, D. L. (2016) Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties. J. Biol. Chem. 291, 11657– 11675,  DOI: 10.1074/jbc.M115.688218
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      Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties
      Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.; Hay, Debbie L.
      Journal of Biological Chemistry (2016), 291 (22), 11657-11675CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), resp. These different CLR-RAMP interactions yield discrete receptor pharmacol. and physiol. effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the mol. details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and detd. the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent mol. modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability anal. indicated unique features that could be used to develop selective small mol. ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.
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      Balo, A. R., Wang, M., and Ernst, O. P. (2017) Accessible virtual reality of biomolecular structural models using the Autodesk Molecule Viewer. Nat. Methods 14, 1122– 1123,  DOI: 10.1038/nmeth.4506
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      Accessible virtual reality of biomolecular structural models using the Autodesk Molecule Viewer
      Balo, Aidin R.; Wang, Merry; Ernst, Oliver P.
      Nature Methods (2017), 14 (12), 1122-1123CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      There is no expanded citation for this reference.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWhtb7L&md5=ff8d429d3b06dd25047f5fa567c44d4a
    40. 40
      Barwell, J., Conner, A., and Poyner, D. R. (2011) Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim. Biophys. Acta, Mol. Cell Res. 1813, 1906– 1916,  DOI: 10.1016/j.bbamcr.2011.06.005
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      40
      Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function
      Barwell, James; Conner, Alex; Poyner, David R.
      Biochimica et Biophysica Acta, Molecular Cell Research (2011), 1813 (10), 1906-1916CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-assocd. transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which assocs. with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP prodn. and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP prodn.; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a no. of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP prodn. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.
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    41. 41
      Barwell, J., Miller, P. S., Donnelly, D., and Poyner, D. R. (2010) Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor. Peptides 31, 170– 176,  DOI: 10.1016/j.peptides.2009.10.021
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      41
      Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor
      Barwell, James; Miller, Philip S.; Donnelly, Dan; Poyner, David R.
      Peptides (New York, NY, United States) (2010), 31 (1), 170-176CODEN: PPTDD5; ISSN:0196-9781. (Elsevier)
      The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examd. by an alanine scan and the results were interpreted with the help of a mol. model. The potency of CGRP at stimulating cAMP prodn. was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a sep. cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can assoc. with RAMPs.
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    42. 42
      Hoang, H. N., Song, K., Hill, T. A., Derksen, D. R., Edmonds, D. J., Kok, W. M., Limberakis, C., Liras, S., Loria, P. M., Mascitti, V., Mathiowetz, A. M., Mitchell, J. M., Piotrowski, D. W., Price, D. A., Stanton, R. V., Suen, J. Y., Withka, J. M., Griffith, D. A., and Fairlie, D. P. (2015) Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists. J. Med. Chem. 58, 4080– 4085,  DOI: 10.1021/acs.jmedchem.5b00166
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      42
      Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists
      Hoang, Huy N.; Song, Kun; Hill, Timothy A.; Derksen, David R.; Edmonds, David J.; Kok, W. Mei; Limberakis, Chris; Liras, Spiros; Loria, Paula M.; Mascitti, Vincent; Mathiowetz, Alan M.; Mitchell, Justin M.; Piotrowski, David W.; Price, David A.; Stanton, Robert V.; Suen, Jacky Y.; Withka, Jane M.; Griffith, David A.; Fairlie, David P.
      Journal of Medicinal Chemistry (2015), 58 (9), 4080-4085CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Cyclic constraints are incorporated into an 11-residue analog of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concns. in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small mol. agonists of the GLP-1 receptor to treat type 2 diabetes.
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    43. 43
      Dal Maso, E., Zhu, Y., Pham, V., Reynolds, C. A., Deganutti, G., Hick, C. A., Yang, D., Christopoulos, A., Hay, D. L., Wang, M. W., Sexton, P. M., Furness, S. G. B., and Wootten, D. (2018) Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy. Biochem. Pharmacol. 150, 214– 244,  DOI: 10.1016/j.bcp.2018.02.005
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      43
      Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy
      Dal Maso, Emma; Zhu, Yue; Pham, Vi; Reynolds, Christopher A.; Deganutti, Giuseppe; Hick, Caroline A.; Yang, Dehua; Christopoulos, Arthur; Hay, Debbie L.; Wang, Ming-Wei; Sexton, Patrick M.; Furness, Sebastian G. B.; Wootten, Denise
      Biochemical Pharmacology (Amsterdam, Netherlands) (2018), 150 (), 214-244CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
      Class B peptide hormone GPCRs are targets for the treatment of major chronic disease. Peptide ligands of these receptors display biased agonism and this may provide future therapeutic advantage. Recent active structures of the calcitonin (CT) and glucagon-like peptide-1 (GLP-1) receptors reveal distinct engagement of peptides with extracellular loops (ECLs) 2 and 3, and mutagenesis of the GLP-1R has implicated these loops in dynamics of receptor activation. In the current study, we have mutated ECLs 2 and 3 of the human CT receptor (CTR), to interrogate receptor expression, peptide affinity and efficacy. Integration of these data with insights from the CTR and GLP-1R active structures, revealed marked diversity in mechanisms of peptide engagement and receptor activation between the CTR and GLP-1R. While the CTR ECL2 played a key role in conformational propagation linked to Gs/cAMP signalling this was mechanistically distinct from that of GLP-1R ECL2. Moreover, ECL3 was a hot-spot for distinct ligand- and pathway-specific effects, and this has implications for the future design of biased agonists of class B GPCRs.
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    44. 44
      Wootten, D., Reynolds, C. A., Smith, K. J., Mobarec, J. C., Koole, C., Savage, E. E., Pabreja, K., Simms, J., Sridhar, R., Furness, S. G., Liu, M., Thompson, P. E., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2016) The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism,. Cell 165, 1632– 1643,  DOI: 10.1016/j.cell.2016.05.023
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      Gkountelias, K., Tselios, T., Venihaki, M., Deraos, G., Lazaridis, I., Rassouli, O., Gravanis, A., and Liapakis, G. (2009) Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding. Mol. Pharmacol. 75, 793– 800,  DOI: 10.1124/mol.108.052423
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      There is no corresponding record for this reference.
    46. 46
      Hollenstein, K., Kean, J., Bortolato, A., Cheng, R. K., Dore, A. S., Jazayeri, A., Cooke, R. M., Weir, M., and Marshall, F. H. (2013) Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438– 443,  DOI: 10.1038/nature12357
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      46
      Structure of class B GPCR corticotropin-releasing factor receptor 1
      Hollenstein, Kaspar; Kean, James; Bortolato, Andrea; Cheng, Robert K. Y.; Dore, Andrew S.; Jazayeri, Ali; Cooke, Robert M.; Weir, Malcolm; Marshall, Fiona H.
      Nature (London, United Kingdom) (2013), 499 (7459), 438-443CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Structural anal. of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 (CRF1R) is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-mol. antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-mol. drugs for diseases of brain and metab.
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