Endocytosis and trafficking of PRL receptor isoforms

描述（由申请人提供）：配体与许多膜受体结合，启动配体和受体的内化。这一过程已被证明会导致多种潜在的命运，包括受体再循环回到表面，溶酶体或蛋白酶体降解配体和/或受体，导致显示受体的下调，或通过时间和空间调节受体的位置来调节信号传导。因此，内吞作用在短期内是细胞反应性的主要调节因素。我们才刚刚开始了解其他类膜受体调节这些过程的分子事件。不同的受体异构体具有不同的信号传导能力，如催乳素（PRL）受体，在相同的靶细胞上表达，但根据目标和环境的不同，表达水平不同。因此，了解这些过程和对PRL作用的净效应对于理解PRL在生理和病理状态下对其靶点的功能至关重要。我们的数据表明，PRL受体异构体以不同的速率内化，配体结合导致“长”而非“短”PRLR异构体的下调，PRL诱导的c-Src活化对“长”而非“短”PRLR异构体的内化很重要。我们假设配体与“长”和“短”PRLR异构体结合导致受体内吞作用和加工的不同机制由细胞质域中的序列决定，这些事件都是由转导信号决定的，并调节净输出。我们建议：1。使用共聚焦显微镜、显性阴性结构体和生化分离比较每个PRLR亚型的内吞途径，并确定负责PRL受体的结构域。2. 使用选择性抑制剂、生化和免疫组织化学方法检查内化后的受体亚型在组成型和配体诱导的内吞作用中的分类和命运，并确定参与的PRL受体结构域。3. 表征同种异构体内化和运输对信号转导的要求，并确定内吞作用与信号转导的关系，通过几种途径。4. 利用嵌合受体和PRL拮抗剂确定二聚化在这一过程中的作用，以及同性与异性二聚化的影响。

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.