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Article

NMR Structure of the Second Intracellular Loop of the α2A Adrenergic Receptor: Evidence for a Novel Cytoplasmic Helix^†^,^‡

Supporting Info

Duane A. Chung,^§ Erik R. P. Zuiderweg,*^§^# Carol B. Fowler, Orkun S. Soyer,^# Henry I. Mosberg,^§ and Richard R. Neubig*^§

Biophysics Research Division and Departments of Pharmacology, Biological Chemistry, Chemistry, Medicinal Chemistry, and Internal Medicine/Hypertension, The University of Michigan, Ann Arbor, Michigan 48109

Biochemistry, 2002, 41 (11), pp 3596–3604

DOI: 10.1021/bi015811+

Publication Date (Web): February 21, 2002

†

This work was supported by NIH Grants HL46417 and GM59462 (R.R.N.), NIH Grant DA03910 (H.I.M.), and by a National Sciences and Engineering Research Council of Canada Postgraduate Scholarship (APP 207830-1998) (D.A.C.). E.R.P.Z. was partially supported by NIH Grant GM52421.

‡

The atomic coordinates for T3-I2 and T3-I2(D130I) (code 1HLL) were deposited in the Protein DataBank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). The chemical shifts were deposited in the BioMagResBank [accession codes 5143 (wild-type T3-I2), 5144 (Rho-T3-I2-T4), 5149 (mutant T3-I2(D130I)), and 5150 (T3-I2-T4)].

Biophysics Research Division.

Department of Pharmacology.

To whom correspondence should be addressed. R.R.N.: Department of Pharmacology, The University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Phone: (734) 763-3650. Fax: (734) 763-4450. E-mail: RNeubig@med.umich.edu. E.R.P.Z.: Biophysics Research Division, The University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109. Phone: (734) 936-3850. Fax: (734) 764-3323. E-mail: zuiderwe@umich.edu.

Department of Biological Chemistry.

Department of Chemistry.

Department of Medicinal Chemistry.

Department of Internal Medicine/Hypertension.

Abstract

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR−G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on 35-residue-long peptides including the critical second intracellular loop (i2) of the α2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4−12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an α2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the α2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.