Characterization of the Hepatic Glucagon Receptor

Abstract

The hepatic glucagon receptor was covalently labeled with [¹²⁵I-Tyr¹⁰]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccini-midyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradio-gram of the gel showed one band at M_r=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 S_{20, w} = 4.3 ± 0.1, Stokes radius = 6.3 ± 0.1 nm, frictional coefficient f/f° = 1.8 and a calculated M_r = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a M_r = 24,000 fragment that binds hormone in a GTP-sensitive manner. The M_r = 24,000 fragment is contained within the M_r = 33,000 fragment. The M_r = 63,000 receptor upon treatment with endo-β-N-acetylglucosamine F for 4 h yields four fragments of apparent M_r = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither M_r = 33,000 and 24,000 fragment appear to be substrates for endo-β-N-acetylglucosaminidase F.

These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of ∼ 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 ∼ 21,000 daltons that does not contain any N-linked glycans.