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J Biol Chem, Vol. 274, Issue 8, 4778-4785, February 19, 1999

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

Maoqing Dong, Xi-Qin Ding, Delia I. Pinon, Elizabeth M. Hadac, Robert P. Oda^§, James P. Landers^¶, and Laurence J. Miller

From the  Center for Basic Research in Digestive Diseases, ^§ Protein Core Facility, Mayo Clinic, Rochester, Minnesota 55905, and ^¶ Analytical Division, Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

The molecular basis of ligand binding to receptors provides important insights 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 resolution separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist 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 fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10¹⁸ mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site 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 the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.

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