B-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

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The goals of this proposal are to characterize the Beta-adrenergic receptors from cultured murine lymphoma cells, the S49 wild type (WT) and the adenylate cyclase-deficient variant cyc-, after the following treatments: (i) epinephrine-induced desensitization and down-regulation of intact lymphoma cells, including studies designed to determine the role of phosphorylation and reversible disulfide bond reduction, (ii) alkylation with N-ethylmaleimide and reduction with dithiothreitol, (iii) inhibition of glycoprotein synthesis with funicamycin, (iv) fractionation of the receptors from epinephrine-treated and control cells on sucrose gradients, and (v) partial proteolytic digestion (peptide mapping). Methodology will include three assays of the Beta-receptor: (i) photoaffinity labeling with [125I] iodoazidobenzylpindolol (IABP) which labels two polypeptides tentatively identified as Beta-receptor polypeptides, (ii) epinephrine-stimulated adenylate cyclase activity, and (iii) binding with labeled Beta-adrenergic receptor antagonist. These approaches should provide insight into the structure of the receptor and help assess the validity of our current concept of receptor desensitization. For example, are the two IABP-labeled polypeptides subunits of an oligomeric receptor; are they related as precursor-product; are they both coupled directly to the other components of the adenylate cylase complex; are both polypeptides simultaneously or sequentially modified during acute or chronic desensitization, and if so, does the mechanism involve either phosphorylation or a reversible reduction-oxidation of disulfide bonds? Knowledge of the structure and mechanisms of Beta-adrenergic receptor desensitization could lead to the more rational use of Beta-receptor agonists and antagonists in the treatment of various diseases such as asthma and high blood pressure.