Abstract

The gastrin-releasing peptide receptor (GRP-R) is a G protein-coupled receptor that mediates a variety of cellular responses, including cell growth and modulation of neuronal activity by activation of heterotrimeric GTP-binding proteins in the Gq family. To understand the regulation of GRP-R signaling we have substituted alanine for each of 10 amino acid residues within the transmembrane (TM) helices of the GRP-R predicted to project into the binding pocket of the receptor and analyzed the importance of each of these residues for receptor function. Two mutations showed selective loss of either agonist (Y285A) or antagonist (F313A) affinity for the GRP-R. In addition, we identified two amino acid residues, Phe²⁷⁰ and Asn²⁸¹, in the sixth TM segment, which are important for receptor-G protein interaction. In a competition-binding assay with an antagonist radioligand, bombesin showed a 20- to 100-fold decreased affinity for the N281A and F270A mutant GRP-R compared with wild-type GRP-R. The saturation-binding isotherms are best fit by a two-state model, indicating that the receptors are in either a low-affinity (K_D2) or a high-affinity (K_D1) state. The ratio of the two affinities (K_D2/K_D1) was significantly increased for both mutants compared with wild-type GRP-R, whereas the fraction of mutant receptors in the high-affinity state (R₁) was decreased. GDP/guanosine-5′-O-(3-thio)triphosphate exchange catalyzed by the N281A mutant was lower than that observed for the wild-type GRP-R. However, for both mutants, bombesin was still able to stimulate 1,4,5-inositol triphosphate in transfected cells albeit with reduced activity. We conclude that these two TM residues are important for receptor-G protein coupling, and postulate that each mutation may affect GRP-R conformational change to the high-affinity, G protein-coupled state.