Chemical modifications of melatonin receptors in chicken brain.
The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for 125I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl (N-ethylmaleimide and p-chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited 125I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of 125I-melatonin binding to guanine nucleotides after N-ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N-acetylimidazole, and p-nitrobenzenesulfonyl fluoride also inhibited 125I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited 125I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.
Institute of Physiology, Academy of Sciences of Czech Republic, Prague.
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Kosar
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Teisinger
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Vyskocil
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J
Vanĕcek
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D008024:Ligands; D008297:Male; D008550:Melatonin / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D044094:Receptors, Melatonin; D013477:Superior Colliculi / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D008024:Ligands; D011956:Receptors, Cell Surface; D044094:Receptors, Melatonin; D014443:Tyrosine; D006639:Histidine; D020245:p-Chloromercuribenzoic Acid; D004228:Dithionitrobenzoic Acid; D019204:GTP-Binding Proteins; D009327:4-Chloro-7-nitrobenzofurazan; D008550:Melatonin; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide