ENDOSOME SORTING OF HUMAN BETA2-ADRENERGIC RECEPTORS

人 β2-肾上腺素能受体的内体分选

• 批准号: 6727705
• 负责人: Robert H Moore
• 金额: $ 28.6万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727705
• 关键词: G protein coupled receptor kinase; adenylate cyclase; asthma; beta adrenergic agent; beta adrenergic receptor; cell line; fluorescence microscopy; immunofluorescence technique; intracellular transport; phosphorylation; protein localization; protein structure function; protein transport; receptor expression; receptor mediated endocytosis; receptor sensitivity

The most commonly used agents in the treatment of acute asthma are drugs that activate human beta2-adrenergic receptors (beta2ARs). Following treatment with agonist, signaling by beta2ARs is greatly reduced by acute and chronic desensitization processes involving several distinct, but overlapping mechanisms - rapid phosphorylation by G- protein coupled receptor kinases (GRKs) and protein kinases A and C, receptor endocytosis into endosomes, and downregulation. Downregulation, which results in a decline in total cellular receptors and lowered sensitivity to beta-agonists, occurs with prolonged exposures to agonist and involves the sorting of receptors to multiple cellular destinations. Our long-range research goal is to understand the mechanisms of endosome sorting of beta2ARs and how these sorting steps determine receptor levels. The objective of the research proposed in this application is to define the role of receptor phosphorylation as a determinant of the intracellular targeting of beta2ARs to the cell surface, to the pericentriolar recycling compartment (PCRC), and to lysosomes. The central hypothesis is that sorting of beta2ARs to the recycling or degradative pathway is regulated by distinct phosphorylation steps and that these steps are key determinants of beta2AR levels. The rationale for the proposed studies is that once the regulation of beta2AR targeting is understood, therapeutic strategies can be designed to alter these pathways and increase the level of receptors available for activation. We are uniquely qualified to successfully complete this research given our expertise in the fields of endosome trafficking and receptor structure/function biology and because of the active collaboration amongst the investigators. We expect to test our central hypothesis and achieve the objective of this proposal by pursuing the following three specific aims: 1) Determine the effect of beta2AR traffic through the PCRC during chronic exposures to agonist and the role of this pathway in the efficient resensitization of receptors following downregulation, 2) Establish GRK2 and GRK5 as regulators of beta2AR sorting to the recycling or degradative pathways during chronic exposures to agonist, and 3) Identify distinct phosphorylation sites within the cytoplasmic tail of the beta2AR that target receptors to the recycling or degradative pathways. In this proposal, we will employ innovative approaches such as inducible expression systems to predictably manipulate the levels of rab11 and GRK and restoration microscopy to define beta2AR subcellular localization with high resolution. Further, we will determine the role in chronic desensitization of specific serine residues in the receptor's cytoplasmic tail that we have recently identified as being essential for acute receptor desensitization. We expect to fully define the role of phosphorylation in determining the intracellular destinations of beta2ARs and to identify how sorting to the PCRC effects receptor function. These results will be significant because they are expected to provide new targets for therapeutic interventions to increase beta2AR levels and thereby improve beta-agonist responsiveness in patients suffering from acute asthma.

治疗急性哮喘最常用的药物是激活人类β2-肾上腺素受体（β2AR）的药物。用激动剂治疗后，β2AR 的信号传导因急性和慢性脱敏过程而大大减少，涉及几种不同但重叠的机制 - G 蛋白偶联受体激酶 (GRK) 和蛋白激酶 A 和 C 的快速磷酸化、受体内吞到内体中，以及下调。下调会导致细胞受体总数下降并降低对 β 激动剂的敏感性，这种下调会在长期暴露于激动剂时发生，并涉及将受体分类到多个细胞目的地。我们的长期研究目标是了解 β2AR 的内体分选机制以及这些分选步骤如何决定受体水平。本申请提出的研究目的是确定受体磷酸化作为β2AR细胞内靶向细胞表面、中心粒周循环室(PCRC)和溶酶体的决定因素的作用。中心假设是，β2AR 的回收或降解途径的分类是由不同的磷酸化步骤调节的，并且这些步骤是 β2AR 水平的关键决定因素。拟议研究的基本原理是，一旦了解了β2AR靶向的调节，就可以设计治疗策略来改变这些途径并增加可激活的受体水平。鉴于我们在内体运输和受体结构/功能生物学领域的专业知识以及研究人员之间的积极合作，我们拥有成功完成这项研究的独特资格。我们期望通过追求以下三个具体目标来检验我们的中心假设并实现本提案的目标：1）确定在长期暴露于激动剂期间通过PCRC的β2AR运输的影响以及该途径在受体的有效再敏化中的作用下调后，2) 建立 GRK2 和 GRK5 作为 β2AR 在长期暴露于激动剂期间循环或降解途径的调节因子，以及 3) 识别β2AR 内不同的磷酸化位点beta2AR 的细胞质尾部将受体靶向回收或降解途径。在本提案中，我们将采用创新方法，例如诱导表达系统来可预测地操纵 rab11 和 GRK 的水平，并采用恢复显微镜来定义高分辨率的 beta2AR 亚细胞定位。此外，我们将确定受体细胞质尾部特定丝氨酸残基在慢性脱敏中的作用，我们最近发现这些残基对于急性受体脱敏至关重要。我们期望充分定义磷酸化在确定 β2AR 细胞内目的地中的作用，并确定 PCRC 分选如何影响受体功能。这些结果意义重大，因为它们有望为治疗干预提供新的靶点，以提高β2AR水平，从而改善急性哮喘患者的β受体激动剂反应性。