CHEMICAL MODIFICATION OF CHOLECYSTOKININ-A RECEPTORS IN RAT PANCREATIC MEMBRANES

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 [H-3]N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)1H-indole-2-carboxamide [(+/-)-[H-3]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 (+/-)-[H-3]-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.