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.
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.
Laboratory of Molecular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, USA.
P J
PJ
Donohue
E
E
Sainz
M
M
Akeson
G S
GS
Kroog
S A
SA
Mantey
J F
JF
Battey
R T
RT
Jensen
J K
JK
Northup
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002999:Clone Cells; D005110:Extracellular Space / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D018004:Receptors, Bombesin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D010446:Peptide Fragments; D018004:Receptors, Bombesin; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001120:Arginine; D019204:GTP-Binding Proteins