title: Structural characterization of Y1 and Y2 receptors for neuropeptide Y and peptide YY by affinity cross-linking.
abstract: Pharmacological studies indicate that peptide YY (PYY) and neuropeptide Y interact with multiple binding sites, categorized as Y1 and Y2 subtypes. In order to identify and structurally characterize the Y1 and Y2 receptors we covalently cross-linked [125I-Tyr36]PYY to its receptors. The Y2 receptor in rat hippocampus and rabbit kidney membranes was affinity labeled using different homo- and heterobifunctional cross-linking reagents. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography resulted in a major labeled protein band of Mr = 50,000 in both hippocampal and kidney membranes, which was unaffected by reducing agents. The Y1 receptor was analyzed in membranes from the MC-IXC human neuroblastoma cell line. Autoradiography revealed two labeled bands at Mr = 70,000 and 45,000. As the intensity of the Mr = 45,000 band was reduced by protease inhibitors, it is likely that this band is a degradation product of the larger band. Labeling of these proteins was obtained only when N-5-azido-2-nitrobenzoyloxysuccinimide was employed for cross-linking followed by exposure to UV light. Labeling of the two cross-linked bands was unaffected by reducing agents. The binding of radiolabeled PYY and the intensity of the cross-linked bands, for both the Y1 and Y2 receptors, were inhibited similarly in a dose-dependent manner by increasing concentrations of unlabeled PYY. When exposed to agarose-coupled lectins, the detergent-solubilized Y1 receptor-hormone complex was completely adsorbed by wheat germ agglutinin and partially by ricin communis II. The cross-linked Y2 receptor was almost totally adsorbed by wheat germ agglutinin-agarose and partially adsorbed by concanavalin A. The adsorptions were in all cases blocked by the appropriate hapten sugar. These results indicate that the Y1 receptor is a glycoprotein with a Mr = 70,000 binding subunit, whereas the Y2 receptor is a glycoprotein with a Mr = 50,000 binding subunit. These results provide evidence that the Y1 and Y2 subtypes of neuropeptide Y and PYY receptors, previously characterized pharmacologically, are structurally distinct glycoproteins, not disulfide-linked to other subunits.
authors.affiliation: Department of Physiology, University of Michigan, Ann Arbor 48109.
authors.forename: S P
authors.initials: SP
authors.lastname: Sheikh
authors.forename: J A
authors.initials: JA
authors.lastname: Williams
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001386:Azides; D002462:Cell Membrane / Q000032:analysis; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006624:Hippocampus / Q000032:analysis*; D007457:Iodine Radioisotopes; D007668:Kidney / Q000032:analysis*; D008562:Membrane Glycoproteins / Q000032:analysis; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D019894:Peptide YY; D010455:Peptides / Q000378:metabolism; D010777:Photochemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000032:analysis* / Q000378:metabolism; D013388:Succinimides
chemical_list: D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D011480:Protease Inhibitors; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D013388:Succinimides; D019894:Peptide YY; C049915:N-hydroxysuccinimidyl-5-azido-2-nitrobenzoate