An Aspartate Residue at the Extracellular Boundary of TMII and an Arginine Residue in TMVII of the Gastrin-Releasing Peptide Receptor Interact To Facilitate Heterotrimeric G Protein Coupling†
- Patrick J. Donohue
- Eduardo Sainz
- Mark Akeson
- Glenn S. Kroog
- Samuel A. Mantey
- James F. Battey
- Robert T. Jensen
- , and
- John K. Northup
The mammalian bombesin receptor subfamily of G protein-coupled receptors currently consists of the gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor, and bombesin receptor subtype 3. All three receptors contain a conserved aspartate residue (D98) at the extracellular boundary of transmembrane domain II and a conserved arginine residue (R309) near the extracellular boundary of transmembrane domain VII. To evaluate the functional role of these residues, site-directed GRP-R mutants were expressed in fibroblasts and assayed for their ability to both bind agonist and catalyze exchange of guanine nucleotides. Alanine substitution at GRP-R position 98 or 309 reduced agonist binding affinity by 24- and 56-fold, respectively, compared to wild-type GRP-R. Single swap GRP-R mutations either resulted in no receptor expression in the membrane (D98R) or the protein was not able to bind agonist (R309D). In contrast, the double swap mutation (D98R/R309D) had high-affinity agonist binding, reduced from wild-type GRP-R by only 6-fold. In situ reconstitution of urea-extracted membranes expressing either wild-type or mutant (D98A or R309A) GRP-R with Gq indicated that alanine substitution greatly reduced G protein catalytic exchange compared to wild-type GRP-R. The D98R/R309D GRP-R had both a higher intrinsic basal activity and a higher overall catalytic exchange activity compared to wild-type; however, the wild-type GRP-R produced a larger agonist-stimulated response relative to the double swap mutant. Taken together, these data show that GRP-R residues D98 and R309 are critical for efficient coupling of GRP-R to Gq. Furthermore, our findings are consistent with a salt bridge interaction between these two polar and oppositely charged amino acids that maintains the proper receptor conformation necessary to interact with G proteins.
This work was supported by NIDCD Grant IRP Z01-DC-00028-02.
Laboratory of Molecular Biology.
These authors contributed equally to this work.
Present address: Department of Vascular Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855.
Present address: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064.
Digestive Disease Branch.
To whom correspondence should be addressed: Director, National Institute on Deafness and Other Communication Disorders, Building 31/Room 3C02, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Telephone: 301-402-0900. Fax: 301-402-1590. E-mail: [email protected].
Laboratory of Cellular Biology.
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