E3 Ligases and Deubiquitinases in GPCR downregulation

描述（由申请人提供）：β 1和β 2肾上腺素能受体是G蛋白偶联受体（gpcr）超家族的成员。该受体超家族包括治疗心血管疾病（如心力衰竭、高血压）和肺部疾病（如哮喘）的一些最重要的药理学靶点。在心力衰竭和哮喘中观察到的肾上腺素能反应衰减的主要原因是受体下调，这是由慢性激动剂暴露引起的。最近，细胞表面受体的泛素化被认为是胞吞后对溶酶体进行分选的重要机制。泛素化是蛋白质的翻译后修饰，是一个明确定义的过程，涉及三种酶活性的级联。其中，由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。我们已经证明两种具有代表性的gpcr， β 2肾上腺素能受体和V2抗利尿激素受体及其衔接蛋白β -抑制素在激动剂刺激下泛素化。β -抑制素的泛素化对于受体内化至关重要，而受体的泛素化对于内化和激活的受体的适当分类和下调至关重要。我们假设泛素化和去泛素化协调了gpcr的运输和信号传导，并涉及一组特定的内吞适配蛋白，包括β -抑制蛋白。具体目的是：1)确定受体泛素化导致受体下调的分子机制；2)确定去泛素化酶在受体运输中的作用。通过泛素化揭示调控gpcr，特别是β - ar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.