Gastric inhibitory polypeptide receptor in hamster pancreatic beta cells. Direct cross-linking, solubilization and characterization as a glycoprotein

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Abstract
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 64000. 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 μM) 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 64000. 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 65000. 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 (a) the GIP receptor in pancreatic beta cells is a protein monomer of apparent Mr 59000; (b) its structure is maintained by intrachain disulfide bridges, these bonds being, however, not involved in the interaction of GIP with its receptor; (c) the GIP receptor is a glycoprotein containing N‐acetylglucosamine, mannose and probably sialic acid in its carbohydrate moiety.