Characterization of covalently cross-linked pancreatic somatostatin receptors.
The receptor for somatostatin present in rat pancreatic plasma membranes was characterized by affinity labeling with [125I-Tyr11]somatostatin utilizing three different heterobifunctional cross-linking agents: N-5-azido-2-nitrobenzoyloxy-succinimide, N-succinimidyl 6-(4-azido 2'-nitrophenylamine)hexanoate, and N-hydroxysuccinimidyl 4-azido-benzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a broad band of Mr = 92,000 when any of the three cross-linkers was used; N-succinimidyl 6-(4-azido 2'-nitrophenylamine), however, was most efficient. Labeling of the Mr = 92,000 protein band was not affected by reducing agents but was sensitive to somatostatin and guanine nucleotides, particularly GTP gamma S, at concentrations which reduced binding to the receptor. The affinity-labeled protein could be solubilized completely with Zwittergent 3-12, partially with Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and poorly with Zwittergent 3-08 and digitonin. When exposed to agarose-coupled lectins, the detergent solubilized, labeled Mr = 92,000 protein was completely adsorbed to wheat germ agglutinin, partially to ricin communis II, and not at all to concanavalin A or lotus or lentil lectin. The Mr = 92,000 protein bound to wheat germ agglutinin-agarose was not eluted by N-acetylglucosamine but was by triacetylchitotriose, providing a considerable purification of the somatostatin receptor. These data allow us to conclude that the somatostatin receptor is a monomeric glycoprotein with an Mr = 90,000 binding subunit which probably contains a polymeric arrangement of N-acetylglucosamine residues.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate