The aim of this project is to purify the hepatic glucagon receptor. The receptor will be solubilized from the membrane with 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propane sulfonate (CHAPS). Binding of [125I]Tyr10-monoiodoglucagon to the solubilized receptor will be measured by the use of the newly developed wheat germ lectin-Sepharose adsorption assay. This assay is based on the glycoprotein nature of the receptor and utilizes the wheat germ lectin-Sepharose as the insoluble matrix to specifically adsorb the hormone receptor complex. The receptor will be purified by chromatographic techniques. These include ion exchange, gel filtration and affinity chromatography over lectin-Sepharose and immobilized glucagon column. Regulation of hormone interaction with the pure receptor by the purified stimulatory regulator of adenylyl cyclase will be studied. Structural correlates of various kinetic states of the receptor will be defined.
