Glucagon is a 29 amino acid pancreatic hormone which counteracts the blood glucose lowering action of insulin by increasing cyclic AMP levels and in turn stimulating hepatic glycogenolysis and gluconeogenesis. Hormone activation of adenylate cyclase requires at least three membrane components; hormone receptor, catalytic subunit and GTP-regulatory component that couples hormone binding to cyclase activation. Isolation and characterization of these components is essential for understanding the mechanism of the sequential events, that begin with hormone binding and end with increase in cyclase activity. We propose to isolate and characterize hepatic glucagon receptor. To this end we have developed procedure of solubilization of liver plasma membrane using digitonin, with retention of glucagon receptor's binding activity. We have also found the 125I-glucagon can be cross-linked to its receptor sites by direct UV irradiation without use of bifunctional reagents. Covalent attachment of glucagon eliminates dissociation of ligand thus provides radioactive marker through out purification procedure. In this proposed investigation, sucrose density gradient centrifugation, iso-electric focusing, and lectin binding procedures will be used to characterize glucagon receptor complex. We also propose to purify glucagon receptor using affinity and high pressure liquid chromatography techniques. Isolation of glucagon receptor and characterization of its molecular properties will lead toward understanding of the mechanisms that control glucose metabolism.
