Beta Adrenergic Receptor Structure and Desensitization

描述(申请人提供)：Beta2-肾上腺素能受体(Beta2AR)是药物开发和分子研究中最具靶向性的七种跨膜G蛋白偶联受体之一，主要是因为哮喘在人群中的流行，其症状可以用专门激活导致支气管扩张的受体的药物来治疗。急性药物刺激细胞内的β2AR会导致快速脱敏(分钟)和慢性刺激(天)，甚至进一步失去其活性，从而降低治疗效果。在过去的几十年里，我们和其他人已经表明，激动剂诱导的快速脱敏机制明显涉及cAMP依赖的蛋白激酶(PKA)和G蛋白受体激酶(GRK)对β2AR的磷酸化、arrestin结合和β2AR从细胞表面到内体的运输。这种缓慢的下调部分与快速的磷酸化事件有关，但也涉及β2AR的蛋白分解和抑制β2AR的合成。先前研究的局限性之一是不能直接测量与β2AR脱敏有关的残基的磷酸化。为了解决这一问题，我们开发了几种针对关键残基的亚磷酸盐特异性抗体，并首次使用它们来直接表征人胚胎肾(HEK)293和表皮样癌A431细胞系中PKA和GRK的磷酸化动力学。在这项建议中，我们打算进一步发展磷酸特异性抗体方法来确定假定的以及已知的GRK和PKA残基磷酸化的动力学。我们将解决以下假设：(I)激动剂以不同的速度刺激单个GRK和PKA位点在Beta2AR上的磷酸化，并且这些相对速率可能会因所使用的特定激动剂而改变；(Ii)参与PKA位点的去磷酸化的磷酸酶在位置和亚型上都不同于GRK位点的去磷酸化；(Iii)多个GRK以位点和细胞类型选择性的方式磷酸化Beta2AR；(Iv)模拟Beta2AR GRK位点的多肽可以成为细胞中GRK磷酸化的有效底物/抑制剂。最后，我们将描述激动剂诱导原代培养的人肺平滑肌细胞β2AR磷酸化、内化、再循环和下调的机制，重点是用于哮喘治疗的激动剂。

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.