Structural characterization of Y1 and Y2 receptors for neuropeptide Y and peptide YY by affinity cross-linking

Journal Article · Tue May 15 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6809865

Sheikh, S P; Williams, J A ^[1]

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1. Univ. of Michigan, Ann Arbor (USA)

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.

• OSTI ID: 6809865
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:14; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
560120 -- Radiation Effects on Biochemicals
Cells
& Tissue Culture
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
CHEMISTRY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DOSE-RESPONSE RELATIONSHIPS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYME INHIBITORS
GLYCOPROTEINS
HIPPOCAMPUS
IMIDES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KIDNEYS
KINETICS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NERVOUS SYSTEM
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PEPTIDES
PHOTOCHEMISTRY
POLYMERIZATION
PROTEINS
RABBITS
RADIOISOTOPES
RATS
REACTION KINETICS
REAGENTS
RECEPTORS
RODENTS
VERTEBRATES