Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[¹²⁵I]iodobenzimidate and N-[4-(4'-azido-3'-[¹²⁵I]iodophenylazo)benzoyl]-3-aminopropionyl-N-oxy-succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2,000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC₅₀= 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using¹²⁵I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monova-lency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone.