Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors. D-Tyr-Gly-((Nle28,31,pNO2-Phe33)CCK-26-33)

S. P. Powers, D. Fourmy, H. Gaisano, L. J. Miller

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

Attempts to biochemically characterize the pancreatic cholecystokinin (CCK) receptor by affinity labeling have utilized either 125I-Bolton-Hunter-CCK-33 ('long' probes) or decapeptide analogues of the carboxyl terminus of CCK ('short' probes), and covalent attachment via the amino-terminal regions of these probes. The long probe has identified a protein of M(r) = 80,000 while 'shorter' probes, which have their site of cross-linking closer to the receptor binding region of the probes, have labeled a distinct protein of M(r) = 85,000-95,000. To extend and complement these observations, we have designed and synthesized a new probe for the CCK receptor which incorporates a photolabile p-nitrophenylalanine moiety within the theoretical receptor-binding region of the hormone, as its carboxyl-terminal residue. This 'intrinsic' photoaffinity labeling probe has been shown to possess full biological activity, with potency and efficacy in stimulating amylase secretion bvy dispersed rat pancreatic acini similar to that of CCK-8 (CCK-26-33). When iodinated oxidatively, this probe binds rapidly in a temperature-dependent, reversible, saturable, specific, high affinity manner to enriched pancreatic plasma membranes. In this work, we have used this probe to specifically label the CCK binding site on rat pancreatic plasma membranes. The M(r) = 85,000-95,000 protein previously identified with amino-terminal cross-linking of short probes appears to be the protein labeled with this reagent as well. This provides strong evidence that this pancreatic plasma membrane protein contains the CCK-binding domain of the CCK receptor. This intrinsic photoaffinity labeling probe should be quite useful for the characterization of the active site of this receptor and for other CCK and gastrin receptors in many species.

Original languageEnglish (US)
Pages (from-to)5295-5300
Number of pages6
JournalJournal of Biological Chemistry
Volume263
Issue number11
StatePublished - Jan 1 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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