title: Chemical modification of cholecystokinin-A receptors in rat pancreatic membranes.
abstract: Chemical modification of amino acids was used to probe the molecular structure of the cholecystokinin-A (CCK-A) receptor on rat pancreatic membranes. Radioligand binding studies with [3H]N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3- yl)1H- 2-carboxamide [(+/-)-[3H]L-364,718], a tritiated highly potent CCK-A receptor antagonist, enabled the evaluation of the effects caused by the modifying reagents. The apparent fragility of the receptor protein necessitated the development of a modification procedure without wash and centrifugation steps. Treatment of a concentrated membrane preparation with the group-specific agents N-ethylmaleimide, phenylglyoxal and diethylpyrocarbonate, subsequent dilution and incubation at lower temperatures (20 degrees C instead of the more generally used 37 degrees C) proved successful. All modifiers affected the binding characteristics for both agonists and antagonists considerably. CCK-A receptor coupling to guanosine-nucleotide-binding proteins was substantially diminished upon modification with N-ethylmaleimide and diethylpyrocarbonate, as could be concluded from the effects on the (+/-)-[3H]L-364,718 displacement by the cholecystokinin C-terminal octapeptide (CCK-8). The ligand-binding site was affected by all three reagents, as could be inferred from the specific protection obtained with the CCK-A receptor antagonist, lorglumide. It therefore appears that sulfhydryl, arginyl, and histidyl residues form an essential part of the ligand-binding domain on the CCK-A receptor and that sulfhydryl and histidyl residues are also involved in the signal-transduction pathway.
authors.affiliation: Center for Bio-Pharmaceutical Sciences, Division of Medicinal Chemistry, Leiden, The Netherlands.
authors.forename: A P
authors.initials: AP
authors.lastname: Ijzerman
authors.forename: C T
authors.initials: CT
authors.lastname: Melman
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D008297:Male; D010179:Pancreas / Q000378:metabolism*; D010658:Phenylglyoxal / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011949:Receptors, Cholecystokinin / Q000187:drug effects* / Q000378:metabolism
chemical_list: D011949:Receptors, Cholecystokinin; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D005033:Ethylmaleimide