BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

肾上腺素刺激对哺乳动物细胞过程的调节 β2肾上腺素能受体(BetaAR)是一种广泛的受体，包括重要的 对神经、心血管和肺部系统的影响。BetaAR 拮抗剂被用来治疗几种心血管疾病 系统，如高血压，和β-AR激动剂是 治疗哮喘。 制约BetaAR有效性的最重要因素之一 激动剂是耐受性的发展速度，被认为是 部分原因是BetaAR的脱敏。脱敏， 腺酰环化酶(AC)的β-AR刺激丢失似乎是导致 通过不同的蛋白激酶使β-AR磷酸化，通过 内部化或下调监管。我们的长期目标是确定 这些机制在调节β-AR反应性中所起的作用 在哺乳动物组织中。 这项建议的主要目标是确定特定的氨基酸在 响应于激活的刺激而被磷酸化的β-AR PKA、PKC或BetaARK介导的在体动物脱敏机制 人胚胎肾(HEK 293)细胞及其后果 磷酸化对ACβ受体激活的影响。主要的焦点将是 推测的BetaARK共有的磷酸化位点。站点定向 β-AR和β-AR中建议的蛋白激酶共同位点的突变 突变对β-氨基丁酸刺激AC影响的研究 随后在HEK 293细胞中的表达将定位所涉及的结构域 在激酶介导的脱敏反应中。这一分析将与 高纯度β-AR的肽谱和显微测序 磷酸多肽用于测定被磷酸化的特定氨基酸 各种不同的激酶。用于肽图谱的β-AR的纯化将 基于血凝素和6-组氨酸表位的使用，这些表位具有 已被引入BetaAR。 次要目标包括：分析BetaArk和 同源脱敏中的内化及其相互依赖； 通过定点突变探索内化结构域； C-末端PKA/PKC共识位点作用的研究 我们提出的部分激动剂导致同源性较低的建议 脱敏作用优于完全激动剂；以及准确的定量 受体数目对脱敏的影响。

The regulation of cellular processes in mammals by epinephrine stimulation of beta2-adrenergic receptors (betaAR) is extensive and includes important effects on the nervous, cardiovascular, and pulmonary systems. BetaAR antagonists are used as therapy for several diseases of the cardiovascular system such as hypertension, and betaAR agonists are a mainstay in the treatment of asthma. One of the most important factors that govern the effectiveness of betaAR agonists is the rate of development of tolerance, which is thought to be caused in part by desensitization of the betaAR. Desensitization, the loss of betaAR stimulation of adenylyl cyclase (AC), appears to be caused by phosphorylation of the betaAR by various protein kinases, by internalization or by downregulation. Our long-term goal is to determine the roles these mechanisms play in the regulation of betaAR responsivity in mammalian tissue. The major goal of this proposal is to identify the specific amino acids in the betaAR that are phosphorylated in response to stimuli that activate PKA-, PKC- or betaARK-mediated mechanisms of desensitization in intact human embryonic kidney (HEK 293) cells, and the consequences of the phosphorylations on betaAR activation of AC. The primary focus will be on the putative betaARK consensus phosphorylation sites. Site-directed mutagenesis of proposed protein kinase consensus sites in the betaAR and the characterization of the mutations' effects on betaAR stimulation of AC following expression in HEK 293 cells will localize the domains involved in the kinase-mediated desensitizations. This analysis will be coupled with peptide mapping and microsequencing of highly purified betaAR phosphopeptides to determine the specific amino acids phosphorylated by the various kinases. Purification of the betaAR for peptide mapping will be based on the use of hemagglutinin and 6-histidine epitopes which have been introduced into the betaAR. Secondary aims include: analysis of the relative roles of betaARK and internalization in homologous desensitization and their interdependence; exploration of internalization domains by site-directed mutagenesis; investigation of the role of the C-terminal PKA/PKC consensus site; tests of our proposal that partial agonists induce less homologous desensitization than do full agonists; and an exact quantitation of the effect of receptor number on desensitization.