Binding sites and transduction process of the cholecystokininB receptor: involvement of highly conserved aromatic residues of the transmembrane domains evidenced by site-directed mutagenesis.

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 CCKB 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 CCKB antagonist L-365,260. However, the addition of saturating CCK8 concentrations to cells expressing this mutant did not result in the production of inositol phosphates, demonstrating the critical role of Phe347 in CCKB receptor to G protein coupling. Substitution of Phe227 with alanine was without effect on the affinities of CCKB ligands and on phosphoinositide turnover but modified the affinity of the CCKA antagonist L-364,718. Residue Trp351 located within the CCKB 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 CCKB receptor accounts for the higher EC50 value 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 CCKB receptor, partially overlapping that of antagonists, and gives new insights into the regions involved in the transduction process.