ENDOCYTOSIS AND REGULATION OF BETA-ADRENERGIC RECEPTORS

β-肾上腺素能受体的内吞作用和调节

• 批准号: 6335463
• 负责人: BRIAN J KNOLL
• 金额: $ 9.5万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-07 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6335463
• 关键词: beta adrenergic receptor; cell line; clathrin; confocal scanning microscopy; dynamin; intracellular transport; lysosomes; phosphorylation; receptor binding; receptor expression; receptor mediated endocytosis; receptor sensitivity; site directed mutagenesis; transfection; vesicle /vacuole

The relaxation of airway smooth muscle is governed in part by agonist binding to G-protein coupled Beta-adrenergic receptors, principally of the Beta2-subtype (Beta2AR). Agonist-activated Beta2ARs desensitize within minutes by rapid phosphorylation and endocytosis, then apparently resensitize by dephosphorylation and recycling back to the cell surface. Prolonged agonist exposure of many hours causes a severe attenuation of responsiveness to Beta-agonist, a large part of which is due to a loss of cellular Beta2ARs, termed downregulation. It is important to understand this process because of the widespread use of Beta2-agonist drugs in therapy for asthma and chronic obstructive pulmonary disease, and the possibility that the efficacy of these drugs may be reduced due to the downregulation of Beta2ARs. Our goal in this proposal is to determine how receptor phosphorylation and endocytosis is related to receptor downregulation. It seems likely that distinct structural features of the Beta2AR are involved in determining the extent of downregulation, since this function is affected by some receptor mutations. However, it is unclear what if any role endocytosis plays in receptor downregulation. To approach this question, we will systematically vary the rate of receptor endocytosis by blocking the formation of endocytic coated vesicles with dynamin dominant negative expression. The endosome concentration of receptors will be varied by constructing cell lines expressing different levels of receptors. The effects of these manipulations on receptor degradation and downregulation will be quantitatively examined to test several models of receptor downregulation. Also, because endocytosis of receptor may be required for its resensitization, we will examine receptor phosphorylation under conditions where endocytosis is inhibited. Finally, we will examine the endocytic pathways used by mutant receptors that downregulate but do not apparently undergo rapid internalization, and receptors that do not downregulate despite their rapid internalization. Completion of these aims will move us significantly closer towards our long-term goal of determining the mechanism of Beta2AR downregulation.

气道平滑肌的放松部分由激动剂支配 与G蛋白偶联的β-肾上腺素能受体结合，主要是 beta2-subtype（beta2ar）。 激动剂激活的β2AR脱敏 在几分钟之内通过快速磷酸化和内吞作用，然后显然 通过去磷酸化并将其回收回到细胞表面来汇总。 长时间的激动剂暴露多个小时会导致严重衰减 对β-激动剂的响应能力，其中很大一部分是由于损失 细胞β2AR的下调。 重要的是 由于广泛使用beta2-agenist，了解此过程 用于治疗哮喘和慢性阻塞性肺疾病的药物， 以及这些药物的功效可能会降低的可能性 下调beta2ars。 我们在此提案中的目标是 确定受体磷酸化和内吞作用与 受体下调。 似乎很有可能结构 beta2ar的特征参与确定的程度 下调，因为此功能受某些受体的影响 突变。 但是，目前尚不清楚任何角色内吞作用是否发挥 在受体下调中。 为了解决这个问题，我们将 系统地通过阻止了受体内吞作用的速率 具有动力蛋白显性阴性的内吞涂层囊泡的形成 表达。 受体的内体浓度将因 构建表达不同水平受体的细胞系。 这 这些操纵对受体降解和 下调将进行定量检查以测试多个模型 受体下调。另外，由于受体的内吞作用可能 它是其敏化所必需的，我们将检查受体 在抑制内吞作用的条件下的磷酸化。 最后，我们将检查突变受体使用的内吞途径 下调但显然不会经历快速的内在化， 和受体尽管迅速而不会下调 内部化。 这些目标的完成将使我们显着 更接近我们确定机制的长期目标 beta2ar下调。