Fat Cell β1 -Adrenergic Receptor: Structural Evidence for Existence of Disulfide Bridges Essential for Ligand Binding

Agents that react chemically with sulfhydryl groups of proteins modify the response of adenylate cyclase to stimulation by β-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 β-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 β-mercaptoethanol, agents that reduce disulfide bridges of proteins, results in a loss of binding of β-adrenergic radioligands to the receptor. The specific binding of radioligands to β-adrenergic receptors that are solubilized in digitonin is affected similarly by treatment with disulfide bridge reducing agents. β-Adrenergic receptor purified from rat fat cells and treated with β-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) /. 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 β-mercaptoethanol (0.1-10%) or dithiothreitol (0.1-10 mM) decreases the ability of the receptor to bind β-adrenergic ligands, decreases the electrophoretic mobility of the receptor, and results in receptor peptides migrating with molecular weight ranging from 54000 to 67000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate. Treating purified receptor with N-ethylmaleimide (10 mM) does not alter the electrophoretic mobility of the receptor (Mr 54000) on polyacrylamide gels under nonreducing conditions. These data demonstrate for the first time the existence of intramolecular disulfide bridges in β-adrenergic receptors and suggest that the integrity of these disulfide bridges is essential for the binding of ligands by the receptor.