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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†

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Laboratories of Molecular Biology and Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, and Digestive Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
Cite this: Biochemistry 1999, 38, 29, 9366–9372
Publication Date (Web):July 1, 1999
https://doi.org/10.1021/bi990544h
Copyright © Not subject to U.S. Copyright. Published 1999 American Chemical Society
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    Abstract

    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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