Endocytosis and trafficking of PRL receptor isoforms

描述（由申请人提供）：与许多膜受体结合的配体启动配体和受体的内化。该过程已显示导致多种潜在命运，包括受体再循环回到表面，配体和/或受体被溶酶体或蛋白酶体降解导致所展示受体的下调，或通过时间和空间调节受体的位置来调节信号传导。因此，内吞作用是短期内细胞反应性的主要调节剂。我们才刚刚开始了解其他类型的膜受体调节这些过程的分子事件。具有不同信号传导能力的不同受体亚型，例如催乳素（PRL）受体的亚型，在相同的靶细胞上表达，但取决于靶和环境以不同的水平表达。因此，了解这些过程和对PRL作用的净效应对于了解PRL在生理和病理状态下的靶点功能至关重要。我们的数据表明，PRL受体亚型以不同的速率内化，配体结合导致下调的“长”，但不是“短”的PRLR亚型，和PRL诱导的激活c-Src是重要的“长”，但不是“短”的PRLR亚型的内化。我们假设，配体结合的“长”和“短”的催乳素受体亚型的结果在受体内吞作用和处理由不同的机制决定的序列在胞质结构域，这些事件都是由转导信号，并调制净输出。我们建议：1.使用共聚焦显微镜、显性阴性结构和生化分离比较每种催乳素受体亚型的内吞途径，并确定负责的催乳素受体的结构域。2.使用选择性抑制剂、生物化学和免疫组织化学方法检查内在化后的分选和受体亚型在组成性和配体诱导的内吞作用中的命运，并鉴定参与的PRL受体结构域。3.表征在异构体的内化和运输中对信号转导的要求，并通过几种途径确定内吞作用与信号转导的关系。4.确定二聚化在此过程中的作用，以及使用嵌合受体和PRL拮抗剂的同源与异源二聚化的影响。

DESCRIPTION (provided by applicant): Ligand binding to many membrane receptors initiates internalization of both ligand and receptor. This process has been shown to result in multiple potential fates, including recycling of receptors back to the surface, degradation of ligand and/or receptor by lysosomes or proteasomes resulting in down-regulation of displayed receptor, or modulation of signaling by temporally and spatially regulating the location of the receptor. Endocytosis is therefore a major modulator of cell responsiveness over the short term. We are only beginning to understand the molecular events regulating these processes for other classes of membrane receptors. Different receptor isoforms with distinct signaling capacities, such as those for the prolactin (PRL) receptor, are expressed on the same target cells, but at different levels depending on target and environment. Thus knowledge of these processes and net effect on PRL action is essential to understanding PRL function at its targets in physiologic and pathologic states. Our data demonstrate that PRL receptor isoforms are internalized at different rates, that ligand binding leads to down regulation of the "long" but not the "short" PRLR isoform, and that PRL-induced activation of c-Src is important for internalization of the "long" but not the "short" PRLR isoform. We hypothesize that ligand binding to the "long" and "short" PRLR isoforms results in receptor endocytosis and processing by distinct mechanisms dictated by sequences in the cytoplasmic domain, and that these events both are determined by transduced signals, and modulate net output. We propose to: 1. Compare endocytic pathways for each PRLR isoform using confocal microscopy, dominant negative constructs, and biochemical fractionation, and identify domains of the PRL receptors which are responsible. 2. Examine post-internalization sorting and fate of receptor isoforms in constitutive and ligand-induced endocytosis using selective inhibitors, biochemical and immunohistochemical approaches, and identify PRL receptor domains which participate. 3. Characterize the requirements for signal transduction in internalization and trafficking of the isoforms, and determine the relationship of endocytosis to signal transduction through several pathways. 4. Determine the role of dimerization in this process, and the effect of homo- vs. hetero-dimerization using chimeric receptors and PRL antagonists.