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

Biochemistry. 1999 Jul 20;38(29):9366-72. doi: 10.1021/bi990544h.


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 G(q) 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 G(q). 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Arginine / genetics
  • Arginine / metabolism*
  • Aspartic Acid / genetics
  • Aspartic Acid / metabolism*
  • Catalysis
  • Clone Cells
  • Extracellular Space / metabolism*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Diphosphate / metabolism
  • Ligands
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Binding / genetics
  • Protein Structure, Tertiary
  • Receptors, Bombesin / biosynthesis
  • Receptors, Bombesin / genetics
  • Receptors, Bombesin / metabolism*


  • Ligands
  • Peptide Fragments
  • Receptors, Bombesin
  • Guanosine Diphosphate
  • Aspartic Acid
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Arginine
  • GTP-Binding Proteins