Cholecystokinin and gastrin are not equally sensitive to GTPγS at CCKB receptors: importance of the sulphated tyrosine

We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and cholecystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCK_B receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCK_B receptors were measured by inhibition of [¹²⁵I]Bolton Hunter-CCK-8 (3-[¹²⁵I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTPγS (guanosine 5′-O-(3-thio)triphosphate) or aluminium tetrafluoride (AlF₄⁻). Activation of the G proteins by GTPγS or AlF₄⁻ led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCK_B receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCK_B receptors and their related intracellular events.