Gastric inhibitory polypeptide receptor in hamster pancreatic beta cells. Direct cross-linking, solubilization and characterization as a glycoprotein.
125I-labelled gastric inhibitory polypeptide (125I-GIP) is directly cross-linked to its specific receptor in hamster pancreatic beta cell membranes by using an ultraviolet irradiation procedure. This approach results in the identification of a GIP-protein complex of apparent Mr 64,000. The labelling of this protein species is specific since it is inhibited when incubating the membranes with increasing doses of native GIP (0.1 nM-1 microM) together with 125I-GIP, half-maximal inhibition being elicited by 5 nM peptide. Reduction of the GIP-protein complex by 100 mM dithiothreitol induces a decrease of the electrophoretic mobility of the complex. Alternatively pretreatment of membranes with dithiothreitol (up to 1 M) does not prevent the binding of 125I-GIP to its receptor. When prelabelled membranes are extracted by 0.5% Triton X-100 (v/v) and the extract is layered on a Sephadex G-50 column, a high peak of radioactivity is eluted with the void volume of the column. Treatment of this peak by 10 min ultraviolet irradiation followed by SDS-PAGE leads to identification of a major band of Mr 64,000. When the peak is further layered on Sephacryl S-200 it yields a single peak of radioactivity corresponding to a protein species with a Stokes radius of 3.2 nm and an apparent Mr of 65,000. The solubilized GIP-receptor complex is specifically adsorbed by Sepharose coupled to wheat germ agglutinin and concanavalin A and eluted from these lectins by their respective sugars. In conclusion the GIP receptor in pancreatic beta cells is a protein monomer of apparent Mr 59 000; its structure is maintained by intrachain disulfide bridges, these bonds being, however, not involved in the interaction of GIP with its receptor; the GIP receptor is a glycoprotein containing N-acetylglucosamine, mannose and probably sialic acid in its carbohydrate moiety.
D000327:Adsorption; D000818:Animals; D006224:Cricetinae; D005749:Gastric Inhibitory Polypeptide / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis*; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007515:Islets of Langerhans / Q000032:analysis*; D037102:Lectins / Q000378:metabolism; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis*; D012995:Solubility
D006023:Glycoproteins; D007457:Iodine Radioisotopes; D037102:Lectins; D011964:Receptors, Gastrointestinal Hormone; D011092:Polyethylene Glycols; D005749:Gastric Inhibitory Polypeptide; D017830:Octoxynol; C041086:gastric inhibitory polypeptide receptor