N-Ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase

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Abstract
125I-Glucagon binding to rat liver plasma membranes was composed of high- and low-affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5''-triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p-(chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylate cyclase system. Stimulation of adenylate cyclase with flouride, guanylyl 5''-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylate cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM. As the NEM concentration increased to 5 or 20 mM, stimulation of adenylate cyclase by each reagent diminished, suggesting that N proteins were alkylated. Glucagon stimulation of adenylate cyclase was impaired in fusions of wild-type S49 cells and alkylated liver plasma membranes, suggesting the presence of a thiol in the glucagon receptor that is important for coupling with Ns. These data demonstrate the presence of multiple reactive thiol groups in the glucagon-sensitive adenylate cyclase system.