Use of Photoaffinity Probes Containing Poly(ethylene glycol) Spacers for Topographical Mapping of the Cholecystokinin Receptor Complex

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand ¹²⁵I-d-Tyr-Gly-[(Nle^28,31)CCK-26–33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 Å, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the M_r = 85 000–95 000 glycoprotein with M_r = 42000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 Å, membrane proteins of M_r = 80000 and M_r = 40000 were specifically labeled. The product of endo-ß-N-acetylglucosaminidase F digestion of the M_r = 80000 protein was M_r = 65000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the M_r = 85 000-95 000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of M_r = 80000 and M_r = 40000 may represent noncovalently associated subunits sited within 16.2 Å of the binding domain. This approach of using flexible, defined-length photolabile probes for the topographical mapping of protein complexes and their domains should be applicable to numerous analogous systems.