Fat cell .beta.1-adrenergic receptor: structural evidence for existence of disulfide bridges essential for ligand binding

作者：Cary P. Moxham, Craig C. Malbon · 发表于：Biochemistry · 年份：1985 · DOI：10.1021/bi00343a007 · 被引用次数：74 · 研究领域：Receptor Mechanisms and Signaling、Pharmacological Effects and Assays、Lipid Membrane Structure and Behavior

Agents that react chemically with sulfhydryl groups of proteins modify the response of adenylate cyclase to stimulation by beta-adrenergic agonists. N-Ethylmaleimide, an agent that alkylates sulfhydryl groups, inactivates both the catalytic moiety of adenylate cyclase and the stimulatory, regulatory guanine nucleotide binding protein Ns of rat fat cells but fails to affect binding of antagonists to the beta-adrenergic receptor [Malbon, C. C., Graziano, M. P., & Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260]. Treating membranes of rat fat cells with dithiothreitol or beta-mercaptoethanol, agents that reduce disulfide bridges of proteins, results in a loss of binding of beta-adrenergic radioligands to the receptor. The specific binding of radioligands to beta-adrenergic receptors that are solubilized in digitonin is affected similarly by treatment with disulfide bridge reducing agents. beta-Adrenergic receptor purified from rat fat cells and treated with beta-mercaptoethanol (10%) and then subjected to gel electrophoresis in the presence of sodium dodecyl sulfate migrates as a Mr 67 000 peptide [Cubero, A., & Malbon, C. C. (1984) J. Biol. Chem. 259, 1344-1350]. In the absence of disulfide bridge reducing agents, however, the purified receptor exhibits greater electrophoretic mobility, migrating as a peptide with Mr 54 000. Treating the native form of the purified receptor with beta-mercaptoethanol (0.1-10%) or dithiothreitol (0.1-10 mM) decreases the ability of the recept...