Beta Adrenergic Receptor Structure and Desensitization

描述（由申请人提供）：β 2-肾上腺素能受体（β 2AR）是药物开发和分子研究中靶向最强的七种跨膜G蛋白偶联受体之一，主要是因为哮喘在人群中的流行，其症状用特异性激活受体的药物治疗，导致支气管扩张。细胞中β 2 AR的急性药物刺激导致快速脱敏（分钟）和慢性刺激（天），甚至进一步丧失其活性，从而降低治疗的有效性。在过去的几十年里，我们和其他人已经表明，快速激动剂诱导的脱敏机制显然涉及cAMP依赖性蛋白激酶（PKA）和G蛋白受体激酶（GRK）磷酸化的β 2 AR，arrestin结合，和运输的β 2 AR从细胞表面到内涵体。缓慢下调部分与快速磷酸化事件相关，但也涉及β 2 AR的蛋白水解和β 2 AR合成的抑制。以前的研究的局限性之一是不能直接测量参与β 2 AR脱敏的残基的磷酸化。为了解决这个问题，我们开发了几个磷酸酶特异性抗体的关键残基，并使用它们直接表征PKA和GRK磷酸化的动力学首次在人胚肾（HEK）293和表皮样癌A431细胞系。在这个建议中，我们打算进一步发展磷酸化特异性抗体的方法，以确定推定的以及已知的GRK和PKA残基磷酸化的动力学。我们将讨论以下假设：（1）激动剂以不同的速率刺激β 2 AR上的GRK和PKA位点磷酸化，并且这些相对速率可能会被所使用的特定激动剂改变;（2）参与PKA位点去磷酸化的磷酸酶与GRK位点去磷酸化的磷酸酶在位置和亚型上都不同;（iii）多种GRK以位点和细胞类型选择性方式磷酸化β 2 AR;（iv）模拟β 2 AR GRK位点的肽可以是细胞中GRK磷酸化的有效底物/抑制剂。最后，我们将在人肺平滑肌细胞的原代培养物中描述激动剂诱导的β 2 AR磷酸化、内化、再循环和下调的机制，重点是用于治疗哮喘的激动剂。

DESCRIPTION (provided by applicant): The beta2-adrenergic receptor (beta2AR) is one of the most targeted seven transmembrane G protein-coupled receptors for drug development and for molecular studies, largely because of the prevalence of asthma in the population, the symptoms of which are treated with drugs that specifically activate the receptor leading to brochodilation. Acute drug stimulation of the beta2AR in cells leads to a rapid desensitization (min) and chronic stimulation (days) to even further loss of its activity, thus reducing the effectiveness of therapy. Over the past several decades we and others have shown that the mechanisms of rapid agonist-induced desensitization clearly involve cAMP-dependent protein kinase (PKA) and G protein receptor kinase (GRK) phosphorylation of the beta2AR, arrestin binding, and trafficking of the beta2AR from the cell surface to endosomes. The slow downregulation is in part correlated with the rapid phosphorylation events but also involves proteolysis of the beta2AR and inhibition of beta2AR synthesis. One of the limitations of previous studies was the inability to directly measure the phosphorylations of residues implicated in beta2AR desensitization. To address this we developed several phosphosite-specific antibodies to key residues and used them to directly characterize the kinetics of PKA and GRK phosphorylations for the first time in human embryonic kidney (HEK) 293 and epidermoid carcinoma A431cell lines. In this proposal we intend to further develop the phospho-specific antibody approach to determine the kinetics of putative as well as known GRK and PKA residue phosphorylations. We will address the following hypotheses: (i) that agonists stimulate individual GRK and PKA site phosphorylations on the beta2AR at different rates, and that those relative rates may be altered by the particular agonist used; (ii) that the phosphatases involved in dephosphorylation of PKA sites differ both in locale and subtype from dephosphorylation of GRK sites; (iii) that multiple GRKs phosphorylate the beta2AR in a site- and cell type selective manner; (iv) that peptides that mimic the beta2AR GRK sites can be effective substrates/inhibitors of GRK phosphorylation in cells. Finally we will characterize the mechanisms of agonist-induced beta2AR phosphorylation, internalization, recycling and downregulation in primary cultures of human lung smooth muscle cells, with an emphasis on agonists used for the treatment of asthma.