Basic Amino Acids at the C‐Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin‐B Receptors

Abstract: In common with other Gq protein‐coupled receptors, the third intracellular loop of the cholecystokinin‐B (CCK‐B) receptor contains three basic amino acids (K333/K334/R335) at the C‐terminal segment. To determine the importance of these conserved basic residues in Gq‐protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS‐7 cells and recording Ca2+‐activated Cl− currents from Xenopus oocytes. Site‐directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK‐B receptors were expressed in COS‐7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK‐8) failed to induce inositol phosphate formation in COS‐7 cells and evoke Ca2+‐activated Cl− currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single‐point mutations resulted in a significant reduction in CCK‐8‐induced inositol phosphate formation and CCK‐8‐activated Ca2+‐dependent Cl− currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK‐8‐simulated inositol phosphate formation and the amplitude of CCK‐8‐evoked Ca2+‐dependent Cl− currents. Radioligand‐binding studies showed that the above‐mentioned mutations did not affect the affinity for CCK‐8 and receptor expression level in COS‐7 cells. These findings suggest that basic amino acids at the C‐terminus of the third cytoplasmic loop are required for the signal transduction by CCK‐B receptors.