Characterization of the glucagon receptor and its functional domains using monoclonal antibodies.
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.
Iwanij
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Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108.
Vincent
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002268:Carboxypeptidases; D002462:Cell Membrane / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism*; D015151:Immunoblotting; D007074:Immunoglobulin G; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D009928:Organ Specificity; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000276:immunology / Q000378:metabolism*; D018027:Receptors, Glucagon
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D005934:Glucagon; D002268:Carboxypeptidases; C022633:serine carboxypeptidase