Abstract
Four monoclonal antibodies, designated 4H11, 6E10, 2C5, and 3E9 were prepared against partially purified rat hepatic glucagon receptor. These antibodies were characterized by their ability to recognize the glucagon receptor in target tissues using immunoblot and immunoprecipitation procedures. The antibodies recognized a 62-kDa receptor band in rat liver, kidney, and adipose tissue but not in lung, adrenals, and erythrocytes, indicating a high degree of specificity. These antibodies recognize different antigenic determinants; the 6E10 and 2C5 bind protein epitopes, while 4H11 and 3E9 bind carbohydrate epitopes. Furthermore, proteolytic mapping of the glucagon receptor established that monoclonal antibodies 6E10 and 2C5 recognize different domains of the receptor molecule. These antibodies were used to study the immunochemical similarities among the receptors from different species and to assess the topological location of the ligand-binding site. By combining the techniques of affinity cross-linking, proteolytic mapping, and antibody binding, we have identified the location of the glucagon-binding site near to the COOH-terminal domain of the receptor.
Highlights
Receptor-The differences in species specificity observed between mAbs 6ElO- and 2C5-staining patterns suggested presence of different epitopes. We investigated this possibility using proteolytic cleavage of the glucagon receptor with S. aureus V-8 protease
Since the glucagon receptor has not been purified to homogeneity, we have developed a simple and rapid procedure for the isolation of glucagon receptor suitable for use as an immunogen
We were able to obtain a positive response for each of three separate fusion experiments that were carried out. This high level of success may be due to the immunogenicity of SDS-denatured glucagon receptor
Summary
Y, bacitracin, PMSF, and detergents (Triton X-100, Nonidet P-40) were purchased from Sigma, leupeptin and pepstatin from Vega Biochemicals, Trasylol, from FBA Pharmaceuticals, and Iodine-125 from Amersham. Eluted proteins were concentrated by precipitation as described above and analyzed by SDS-PAGE, isoelectric focusing, and autoradiography This preparation yielded between 100 and 200 fig of partially purified receptor from 30 to 60 mg of plasma membranes. Min washes in the buffer described above but without the protein, the blots were incubated with anti-mouse antibody (1:5,000 dilution) conjugated to alkaline phosphatase. After another series of washes, the bound antibodies were visualized as recommended by the supplier. Zmmunoprecipitation-Affinity cross-linked liver plasma membranes were solubilized with 0.5% octyl glucoside in Tris-HCl buffer, pH 7.4, as described by Bartles et al [23]. Glucagon was iodinated as described by Lin et al [33], and separation of iodinated glucagon from unincorporated iodine was carried out as described by Iwanij and Hur [10]
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