The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor

The hepatic glucagon receptor was covalently labeled with [¹²⁵I-Tyr¹⁰]monoiodoglucagon ([¹²⁵I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the M(r) = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [¹²⁵I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: s(20,w) = 4.3 ± 0.1, Stokes radius = 6.3 ± 0.1 nm, frictional coefficient f/f(o) = 1.8, and a calculated M(r) = 119,000. Incubation of liver membranes at 32° C for 15 min prior to the addition of [¹²⁵I]MIG permitted us to identify the high molecular weight form (M(r) = ~113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The M(r) = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [¹²⁵I]MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [¹²⁵I]MIG binding activity. [¹²⁵I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [¹²⁵I]MIG with the soluble receptor is insensitive to the presence of GTP. IC₅₀ for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC₅₀ in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the M(r) = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.