BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

这项提案的总体目标是阐明该综合体 导致快速脱敏和敏化的机制 β2-肾上腺素能受体(Beta2AR)对腺苷酸环化酶(AC)的刺激作用。 所获得的知识将有助于理解疾病，如 这些机制可能改变的哮喘和高血压，以及在 药理学的基本原理 干预。 这项提案有三个具体目标，重点是确定 磷酸化/去磷酸化在细胞周期调控中的作用 AC对β2受体刺激的反应性。第一个目标是确定 参与cAMP依赖蛋白的β_2AR的精确结构域 蛋白激酶C(PKC)与β肾上腺素能受体 脱敏的激酶途径。将构建Beta2AR突变体， 在L细胞中的表达及其对AC的调节作用 对引起脱敏的各种治疗。要测量 将构建磷酸化、突变体Beta2AR的化学计量比 含有外来表位的目的是为了快速发展 基于表位的纯化回收程序 绑定到地缘列。 第二个目标是评估PKC介导的假设 Gi/α亚基的磷酸化和失活 GTP结合蛋白，或AC的催化亚单位是 4β-佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)和嘌呤能受体- AC的诱导敏化。这将通过使用 免疫沉淀法测定Gi/α亚基的磷酸化 和AC催化亚基，构建这些蛋白质的突变体以 帮助纯化和分析所涉及的结构域，并进行重组 研究以确定胃肠道功能是否可以恢复。PKC同工酶 将确定参与PMA和嘌呤能敏化的情况 通过免疫沉淀和佛波醇测定易位 二丁酸盐结合。 第三个目标是确定参与蛋白质合成的磷酸酶。 对完整细胞的脱敏/增敏作用 磷酸酶抑制剂。

The overall goal of this proposal is the elucidation of the complex mechanisms which cause the rapid desensitization and sensitization of beta2-adrenergic receptor (beta2AR) stimulation of adenylyl cyclase (AC). The knowledge gained will help in the understanding of diseases such as asthma and hypertension in which these mechanisms may be altered, and in the rationale for pharmacological intervention. This proposal has three specific aims which are focused on determining the role of phosphorylation/dephosphorylation in the regulation of the responsivity of AC to beta2AR stimulation. The first aim is to determine the precise domains of the beta2AR involved in the cAMP-dependent protein kinase, the protein kinase C (PKC) and the beta-adrenergic receptor kinase pathways of desensitization. beta2AR mutants will be constructed, expressed in L cells and their regulation of AC characterized in response to various treatments which elicit desensitization. To measure the stoichiometry of phosphorylation, mutant beta2AR's will be constructed containing foreign epitopes with the intent of developing a rapid high recovery procedure for purification based on epitope binding to affinity columns. The second aim is to assess the hypothesis that PKC-mediated phosphorylation and inactivation of either the Gi/alpha subunits of the GTP-binding proteins, or the catalytic subunit of AC is the mechanism of the 4Beta-phorbol 12-myristate 13-acetate (PMA) and purinergic receptor- induced sensitizations of AC. This will be accomplished through the use of immunoprecipitation to measure phosphorylation of Gi/alpha subunits and AC catalytic subunits, construction of mutants of these proteins to aid in purification and analysis of domains involved, and reconstitution studies to determine if Gi function can be restored. PKC isozyme involvement in the PMA and purinergic sensitizations will be identified by measurement of translocation by immunoprecipitations and phorbol dibutyrate binding. The third aim is to identify phosphatases involved in the desensitization/sensitization in intact cells through the use of phosphatase inhibitors.