Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor - Rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis

The molecular basis of ligand binding to receptors provides important insig hts for drug development. Here, we explore domains of the cholecystokinin ( CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high resol ution separation of peptide fragments by capillary electrophoresis, Each pr obe incorporated the same fluorophore and a photolabile p-benzoylphenylalan ine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-3 3) of CCK analogues representing full agonist, partial agonist, and antagon ist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced flu orescence detection achieved an on-capillary mass sensitivity of 1.6 attomo les (10(-18) mol), with an excellent signal to-noise ratio, Each of the bio logically divergent, but structurally similar probes saturably and specific ally labeled the same receptor domain, consistent with conservation of "doc king" determinants. This had an apparent mass of 2.9 kDa, most consistent w ith the first extracellular loop domain. An additional probe having its sit e of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of th e specific receptor residue(s) covalently linked to the amino-terminal prob es must await further fragmentation and sequence analysis.