Endocytosis and trafficking of PRL receptor isoforms

PRL 受体亚型的内吞作用和运输

• 批准号: 7071221
• 负责人: LINDA A. SCHULER
• 金额: $ 21.13万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071221
• 关键词: biological signal transduction; chimeric proteins; confocal scanning microscopy; endocytosis; hormone receptor; immunocytochemistry; prolactin; protein isoforms; protein sequence; protein transport; receptor binding

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.

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在化。这一过程导致了多种潜在的命运，包括受体返回表面，通过溶酶体或蛋白酶体降解配体和/或受体，导致显示的受体下调，或者通过在时间和空间上调节受体的位置来调节信号。因此，内吞作用在短期内是细胞反应性的主要调节器。我们才刚刚开始了解调节其他类型的膜受体这些过程的分子事件。不同的受体亚型具有不同的信号传递能力，例如催乳素受体(PRL)受体的受体亚型在相同的靶细胞上表达，但表达水平取决于靶细胞和环境。因此，了解这些过程和对PRL作用的净影响对于理解PRL在生理和病理状态下靶点的功能是至关重要的。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“长”而不是“短”的PRLR亚型下调，并且PRL诱导的c-Src的激活对“长”而不是“短”的PRLR亚型内化是重要的。我们假设，与“长”和“短”PRLR亚型结合的配体导致受体内吞作用和由细胞质结构域序列决定的不同机制的处理，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、显性负结构和生化分级比较PRLR各亚型的内吞途径，并确定负责的PRL受体结构域。2.利用选择性抑制剂、生化和免疫组织化学方法，检测内化后受体异构体在结构性和配体诱导的内吞作用中的分类和去向，并确定参与其中的PRL受体结构域。3.确定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之间的关系。4.确定二聚化在这一过程中的作用，以及使用嵌合受体和PRL拮抗剂进行同源和异源二聚化的效果。