Characterization of the hepatic glucagon receptor

The hepatic glucagon receptor was covalently labeled with [125I-Tyr10]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccinimidyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradiogram of the gel showed one band at Mr = 63,000 that was sensitive to excess unlabeled glucagon and GTP. 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 S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f degrees = 1.8 and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees for 15 min prior to the addition of [125I-Tyr10] permitted us to identify the high molecular weight form (Mr approximately equal to 113,000) by direct SDS-gel electrophoretic analysis. Limited elastase treatment of the hormone-occupied receptor results in the appearance of a Mr = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a Mr = 24,000 fragment that binds hormone in a GTP-sensitive manner. The Mr = 24,000 fragment is contained within the Mr = 33,000 fragment. The Mr = 63,000 receptor upon treatment with endo-beta-N-acetylglucosamine F for 4 h yields four fragments of apparent Mr = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither Mr = 33,000 and 24,000 fragment appear to be substrates for endo-beta-N-acetylglucosaminidase F. These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of approximately 60,000 dalton hormone binding subunit which is a glycoprotein containing at least four N-linked glycans accounting for 18,000 daltons of its mass. Both the hormone binding function and the capacity for the interaction with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans.