Abstract

The functional significance of the extracellular amino-terminal region and of three highly conserved aromatic residues present in the fifth (TM-V) and sixth (TM-VI) transmembrane domains of the rat cholecystokinin (CCK)_B receptor, transfected in Cos-7 cells, was investigated by site-directed mutagenesis. The amino-terminal region of the CCK_B receptor seemed to be weakly involved in CCK binding in that the affinities of CCK₈ and selective agonists and antagonists were not modified by truncation of this region. Substitution of Phe347 in TM-VI with alanine produced a mutant receptor that displays the same affinity and selectivity as the wild-type receptor for agonists, but a slightly increased affinity for the selective CCK_B antagonist L-365,260. However, the addition of saturating CCK₈concentrations to cells expressing this mutant did not result in the production of inositol phosphates, demonstrating the critical role of Phe347 in CCK_B receptor to G protein coupling. Substitution of Phe227 with alanine was without effect on the affinities of CCK_B ligands and on phosphoinositide turnover but modified the affinity of the CCK_A antagonist L-364,718. Residue Trp351 located within the CCK_B receptor TM-VI is involved in the binding of CCK₈ and CCK₄ and of the CCK₄-based antagonist PD-134,308, as illustrated by the decreased affinities of these ligands in W351A mutant. The lower affinity for CCK₈ observed with this mutated CCK_B receptor accounts for the higher EC₅₀value for phosphotidylinositol hydrolysis. This study suggests that at least part of the binding site for the agonist is located inside the transmembrane domain of the CCK_B receptor, partially overlapping that of antagonists, and gives new insights into the regions involved in the transduction process.