B-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

这项建议的目标是描述β-肾上腺素能 培养的小鼠淋巴瘤细胞、S49野生型(WT)和 腺苷环化酶缺陷型变异体Cyc-，以下为 治疗：(I)肾上腺素诱导的脱敏和下调 完整的淋巴瘤细胞，包括旨在确定 磷酸化和可逆二硫键还原，(Ii)烷基化 N-乙基马来酰亚胺和二硫苏糖醇还原，(Iii)抑制 用Funicamine合成糖蛋白，(Iv) 来自肾上腺素处理细胞和对照细胞的蔗糖梯度受体， 和(V)部分蛋白水解酶(肽图谱)。方法论将 包括三种检测β受体的方法：(I)光亲和标记 [125I]标记两个多肽的碘叠氮苯并吲哚 初步鉴定为β受体多肽，(II) 肾上腺素刺激的腺苷环化酶活性，以及(Iii)与 标记的β肾上腺素能受体拮抗剂。这些方法应该 提供对受体结构的洞察，并帮助评估 我们目前的受体脱敏概念的有效性。例如, 是寡聚受体的两个IABP标记的多肽亚基； 它们是否作为前体产品相关；它们是否都直接耦合到 腺苷环化酶复合体的其他成分；两种都是多肽 在急性或慢性期间同时或顺序修改 脱敏，如果是这样的话，机制是否涉及 是磷酸化还是二硫键的可逆还原氧化？ 对β-肾上腺素能受体结构和机制的认识 脱敏可导致更合理地使用β-受体 激动剂和拮抗剂在治疗各种疾病中的作用 哮喘和高血压。

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.