ENDOCYTOSIS AND REGULATION OF BETA-ADRENERGIC RECEPTORS

气道平滑肌的松弛部分由激动剂控制， 与G蛋白偶联的β-肾上腺素能受体结合，主要是 β 2亚型（Beta2 AR）。 激动剂激活的Beta2 AR脱敏 在几分钟内通过快速磷酸化和内吞作用， 通过去磷酸化和再循环回到细胞表面而重新敏感。 长时间的激动剂暴露数小时会导致 对β-激动剂的反应性，其中很大一部分是由于 称为下调。 重要的是要 由于β 2受体激动剂的广泛使用， 治疗哮喘和慢性阻塞性肺病的药物， 以及这些药物的功效可能由于以下原因而降低的可能性 β 2AR的下调。 我们在这个提案中的目标是 确定受体磷酸化和内吞作用如何与 受体下调 很有可能， Beta2 AR的特征参与确定 下调，因为这种功能受到某些受体的影响， 突变。 然而，目前还不清楚内吞作用是否起作用， 在受体下调中。 为了解决这个问题，我们将 系统地改变受体内吞作用的速率，通过阻断 形成内吞包被囊泡，发动蛋白显性阴性 表情 受体的内体浓度将通过以下方式变化： 构建表达不同水平受体的细胞系。 的 这些操作对受体降解的影响， 下调将被定量检查，以测试几个模型 受体下调。此外，由于受体的内吞作用可能 为了使其重新敏感，我们将检查受体 在胞吞作用被抑制的条件下磷酸化。 最后，我们将研究突变受体使用的内吞途径 下调但没有明显经历快速内化， 和受体，不下调，尽管他们的快速 内化 这些目标的实现将使我们取得重大进展 更接近我们的长期目标，确定机制， β 2AR下调。

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.