Basic amino acids at the C-terminus of the third intracellular loop are required for the activation of phospholipase C by cholecystokinin-B receptors.
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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.
Wang
H L
HL
Department of Physiology, Chang Gung College of Medicine and Technology, Tao-Yuan, Taiwan, R.O.C.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D019556:COS Cells / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004594:Electrophysiology; D004789:Enzyme Activation; D005260:Female; D019204:GTP-Binding Proteins / Q000502:physiology; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D051381:Rats; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D014982:Xenopus laevis
D000596:Amino Acids; D002712:Chlorides; D018076:DNA, Complementary; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium