Affinity labeling of a novel cholecystokinin-binding protein in rat pancreatic plasmalemma using new short probes for the receptor

Previous biochemical characterizations of the cholecystokinin (CCK) receptor have used the 'long' probe ¹²⁵I-Bolton-Hunter-CCK-33 since it was the only CCK analogue with high affinity and high specific radioactivity which possessed an amino group available for chemical cross-linking. These studies have consistently identified a major binding protein of approximately 81 kilodaltons and have identified several minor proteins which were obtained under different cross-linking conditions and in different laboratories. Because the receptor-binding region of CCK-33 (carboxyl-terminal heptapeptide) is so far removed from the radiolabel and from available amino groups (positions 1 and 11), this probe carries potential for proteolytic cleavage of label from receptor and for labeling 'near neighbors' instead of the binding site. We therefore designed two 'short' probes for the CCK receptor. ¹²⁵I-Bolton-Hunter-Lys-Gly-CCK-8 has an ε-amino group available for cross-linking. ¹²⁵I-Tyr-[Thr²⁸,Nle³¹]CCK-25-33 has an α-amino group for cross-linking and has the major advantage of being labeled by oxidative means, unique for CCK derivatives. Both radioiodinated decapeptides were purified by reverse-phase high pressure liquid chromatography to yield specific radioactivity of 2,000 Ci/mmol; demonstrated saturable, specific, and high affinity binding to rat pancreatic plasma membranes; and retained full biological activity to stimulate amylase secretion. Using a variety of cross-linking methods, these probes each identified the same M(r) = 85,000-95,000 protein in rat pancreatic plasmalemma, and CCK-8 competed for this labeling in a concentration-dependent manner (IC₅₀ = 1 nM). No change in apparent mobility of this band was observed under reducing or nonreducing conditions, suggesting lack of covalent attachment to other subunits. The M(r) = 85,000-95,000 species migrated differently on sodium dodecyl sulfate gels than any of the components previously identified using ¹²⁵I-Bolton-Hunter-CCK-33, confirming the novel nature of this binding protein. These short probes should be very useful for further characterization of CCK receptor on this and other tissues.