Ligand association with the rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y (NPY) receptors shows large subtype-related differences in sensitivity to chaotropic and alkylating agents.
The binding to rabbit kidney or hypothalamic particulates of the subtype-selective neuropeptide Y (NPY) receptor ligands [125I](Leu31,Pro34)hPYY (as Y1 site label at 2 nM human pancreatic polypeptide (hPP)), [125I]-hPYY(3-36) (Y2 label), and [125I]-hPP (Y5 label) displayed great differences in sensitivity to alkylators and chaotropic agents. Sensitivity to a nonionic chaotrope, urea, was much higher for the Y1 binding than for the Y5-like binding or the Y2 binding. The non-selective alkylator N-ethylmaleimide (NEM) and several alkylators selective for aminergic receptors were much more efficacious against the Y1 relative to the Y2 binding. Similar differences could be confirmed with the attachment of Y1 and Y2-selective tracers to CHO cells expressing the cloned guinea-pig Y1 or Y2 receptors. The Y5-like binding was quite insensitive to NEM, but sensitive to chloroethylclonidine (CEC) and prazobind, which were less potent at the Y1, and especially at the Y2 site. The unrestricted-access alkylator 2-aminoethyl methanethiosulfonate inhibited the binding to all subtypes, while the restricted-access agent 2-(trimethylammonium)ethylmethanethiosulfonate poorly inhibited the Y5-like binding, or the guanine nucleotide-insensitive Y2 binding. These results are compatible with an active conformation of the Y5-like site dependent on maintenance of a shared hydrophobic cavity. The Y2 sites resistant to guanosine polyphosphates and restricted-access alkylators were detected mainly in particulates slowly solubilized by cholate at 0-5 degrees C; these sites could be clustered.
Parker
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SL
Department of Pharmacology, University of Tennessee College of Medicine, Memphis 38163, USA. slparker@utmem1.utmem.edu
Parker
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MS
D000477:Alkylating Agents / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D003000:Clonidine / Q000031:analogs & derivatives / Q000378:metabolism; D006224:Cricetinae; D003538:Cystamine / Q000031:analogs & derivatives / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D005768:Gastrointestinal Hormones / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008024:Ligands; D008698:Mesylates / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D019894:Peptide YY / Q000378:metabolism*; D011817:Rabbits; D011869:Radioligand Assay; D017476:Receptors, Neuropeptide Y / Q000378:metabolism*; D020358:Sodium Cholate / Q000378:metabolism; D013438:Sulfhydryl Compounds; D014508:Urea / Q000378:metabolism
D000477:Alkylating Agents; D005768:Gastrointestinal Hormones; D008024:Ligands; D008698:Mesylates; D010446:Peptide Fragments; D017476:Receptors, Neuropeptide Y; D013438:Sulfhydryl Compounds; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor; C100568:neuropeptide Y5 receptor; C106655:peptide YY, iodo-Leu(34)-Pro(34)-; D019894:Peptide YY; C061785:peptide YY (3-36); C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C051731:chlorethylclonidine; C029998:benextramine; D000584:Amiloride; D014508:Urea; D019205:GTP-Binding Protein alpha Subunits, Gs; D003000:Clonidine; D020358:Sodium Cholate; D005033:Ethylmaleimide; D003538:Cystamine