E3 Ligases and Deubiquitinases in GPCR downregulation

描述(申请人提供)：β1和β2肾上腺素能受体是G蛋白偶联受体(GPCRs)超家族的成员。这个受体超家族包括一些最重要的治疗心血管疾病(如心力衰竭、高血压)和肺部疾病(如哮喘)的药理靶点。在心力衰竭和哮喘中观察到的肾上腺素能反应减弱的一个主要原因是受体下调，这是慢性激动剂暴露的结果。最近，细胞表面受体的泛素化被认为是内吞后分选溶酶体的重要机制。泛素化是蛋白质的翻译后修饰，由一个明确的过程协调，涉及三种酶活性的级联。其中，酶E3泛素连接酶催化的最后一步决定了底物泛素化的特异性。我们已经证明，两个具有代表性的GPCRs，β2肾上腺素能受体和V2加压素受体及其适配器蛋白β-arrestin在激动剂刺激下变得泛素化。β-arrestin的泛素化是受体内化的关键，而受体的泛素化对于内化和激活的受体的正确分类和下调是必不可少的。我们假设泛素化和去泛素化协调GPCRs的运输和信号传递，并涉及一组特定的内吞适配蛋白，包括β-拦截素。其具体目的是：1)明确受体泛素化导致受体下调的分子机制；2)确定去泛素化酶(S)在受体转运中的作用。解开泛素化调控GPCRs，特别是β-ARs的分子机制，可能对心血管和肺部疾病的新治疗策略的发展产生重大影响。

DESCRIPTION (provided by applicant): The beta 1 and beta 2 adrenergic receptors are members of the super-family of G protein-coupled receptors (GPCRs). This receptor super-family comprises some of the most important pharmacological targets for the treatment of cardiovascular (e.g. heart failure, hypertension) and pulmonary diseases (e.g. asthma). A major cause for the observed attenuation of adrenergic response in heart failure and asthma is receptor downregulation, which results from chronic agonist exposure. Recently, ubiquitination of cell-surface receptors has been implicated as an important mechanism for post-endocytic sorting to lysosomes. Ubiquitination is a post-translational modification of proteins orchestrated by a well-defined process involving a cascade of three enzymatic activities. Of these the final step, catalyzed by the enzyme, E3 ubiquitin ligase, determines the specificity of substrate ubiquitination. We have demonstrated that two representative GPCRs, the beta2 adrenergic receptor and the V2 vasopressin receptor and their adaptor protein beta-arrestin become ubiquitinated upon agonist stimulation. Ubiquitination of beta-arrestin is crucial for receptor internalization, whereas ubiquitination of the receptor is essential for the proper sorting and downregulation of the internalized and activated receptors. We hypothesize that ubiquitination and deubiquitination coordinate the trafficking and signaling of GPCRs and involve a specific set of endocytic adapter proteins including beta-arrestins. The specific aims are: 1) to define the molecular mechanisms of receptor ubiquitination leading to receptor downregulation, and 2) to determine the roles of deubiquitinating enzyme(s) in receptor trafficking. Unraveling the molecular mechanisms governing the regulation of GPCRs, especially beta ARs, by ubiquitination could have a great impact on the development of novel therapeutic strategies for cardiovascular and pulmonary diseases.