Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179

The full peptide antagonist of the pancreatic cholecystokinin (CCK) receptor, JMV 179, [Boc-Tyr(SO3H)-Ahx-Gly-dTrp-Ahx-Asp phenylethyl ester, where Tyr(SO3H) = sulfated tyrosine, Ahx = 6-aminohexanoic acid] was modified at its N-terminus by incorporation of p-hydroxyphenyl propionate (Bolton-Hunter reagent, BH) and was subsequently radioiodinated. After HPLC purification, 125I-BH-JMV-179, a CCK antagonist radioligand of high specific activity (2000 Ci/mmol) was obtained. 125I-BH-JMV-179 bound to a single population of sites on rat pancreatic plasma membranes, (Kd = 3.9 nM, Bmax = 40 pmol/mg protein). Binding was dependent on time, temperature, and protein concentration, and was fully reversible. JMV 179 radioligand detected four times as many sites as an agonist radioligand [C. Hadjiivanova, M. Dufresne, S. Poirot, P. Sozzani, N. Vaysse, L. Moroder and D. Fourmy (1992) Eur. J. Biochem. 204, 273-279]. Agonists and antagonists of the A- and B-subtype CCK/Gastrin receptors inhibited 125I-BH-JMV-179 binding with an order of potency compatible with the A-subtype CCK receptor pharmacology. Moreover, the sulfate group on the tyrosine residue of the CCK Peptides appeared to be of much less importance for antagonist affinity than for agonist affinity. Inhibition of 125I-BH-JMV-179 binding by agonists (except JMV 180), demonstrated the presence of two affinity classes of binding sites. The population of sites having an apparent high affinity for CCK represented 30 pmol/mg protein and threefold the number of high-affinity sites previously identified by an agonist radioligand. In presence of non-hydrolyzable GTP, all the sites bound CCK agonists with a low affinity. Moreover, saturation analysis of JMV 179 radioligand binding in the presence of CCK indicated that CCK interacted competitively with all JMV 179 sites and demonstrated binding of JMV 179 radioligand to two distinct affinity classes of sites. In the presence of GTP[S] a single affinity class of sites for JMV 179 radioligand was found as in the control experiments without CCK. This study, with the first CCK peptide antagonist radioligand, demonstrates that CCK receptors exist in two interconvertible affinity states regulated by guanine-nucleotide-binding regulatory protein(s) in rat pancreatic plasma membranes. JMV 179 radioligand does not induce receptor coupling but distinguishes the two affinity states of the CCK receptors. JMV 179 reveals the existence of populations of high-affinity and low-affinity sites for CCK which had not previously been detected by agonist radioligand binding, thus suggesting heterogeneity of CCK receptor sites in membranes.