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CELL AND MOLECULAR BIOLOGY OF THE TRH RECEPTOR

TRH 受体的细胞和分子生物学

基本信息

批准号：
6380035
负责人：
THOMAS K GRAVES
金额：
$ 7.67万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-01 至 2002-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6380035
关键词：
G protein confocal scanning microscopy endocrine pharmacology endocytosis hormone receptor intracellular transport lysosomes mutant nucleic acid sequence oligonucleotides pituitary gland protein transport receptor binding receptor expression thyrotropin releasing hormone tissue /cell culture vesicle /vacuole

项目摘要

The pituitary receptor for the hypothalamic peptide thyrotropin releasing hormone (TRH) is a G-protein coupled, calcium-mobilizing receptor. In response to agonist binding, the TRH-receptor complex undergoes rapid and extensive internalization. The receptor recycles to the plasma membrane following removal of the ligand over longer times, the receptor undergoes homologous downregulation. We have demonstrated receptor cycling directly using fluorescently labeled TRH agonists to follow the ligand, and using immunocytochemistry to localize epitope-tagged receptors stably expressed in pituitary lactotrophs. The function of this rapid and extensive cycling is completely unknown. We hypothesize that ligand-dependent endocytosis and recycling of the TRH receptor are important in the TRH response; specifically, we will test whether internalization is necessary for desensitization, resensitization, or downregulation of the TRH receptor. We have shown that a mutant TRH receptor with a truncated C- terminus fails to internalize TRH but can generate an increase in intracellular calcium in response to agonists. We will first determine which regions of the TRH receptor are required for ligand-dependent internalization and then develop pituitary cell lines expressing a variety of mutant receptors that are abnormal in internalization or recycling. We will then test the possibility that ligand-dependent internalization is responsible for either the desensitization or resensitization of the TRH response. We have shown that cells display an impaired calcium response to TRH if they are exposed to pulses of TRH spaced close together but a full response to pulses of TRH spaced at least ten minutes apart. We will compare the calcium responses of cells expressing normal or internalization-defective receptors. We will also measure TRH responses following selective inhibition of internalization or recycling. Finally, we hypothesize that while most receptor recycles following internalization, a fraction of the receptors are sorted to a degradative, lysosomal pathway, accounting for gradual homologous downregulation. We will test this hypothesis by measuring the ability of TRH to regulate receptor density in cells expressing internalization- defective receptors, and in cells in which endocytosis and recycling have been selectively inhibited.

下丘脑肽促甲状腺激素释放的垂体受体激素(TRH)是一种G蛋白偶联的钙动员受体。在……里面对激动剂结合的反应，TRH-受体复合体经历快速和广泛的内部化。受体循环至质膜在较长时间移除配体后，受体经历相应的下调监管。我们已经直接演示了受体循环。使用荧光标记的TRH激动剂跟随配体，并使用免疫细胞化学定位稳定表达的表位标记受体在脑下垂体促乳素细胞中。这一功能迅速而广泛骑自行车完全不为人所知。我们假设配基依赖 TRH受体的内吞作用和循环在TRH中起重要作用响应；具体地说，我们将测试是否需要内部化用于TRH的脱敏、再增敏或下调受体。我们已经证明，突变的TRH受体具有截短的C- Terminus无法内化TRH，但会增加细胞内钙对激动剂的反应。我们将首先确定 TRH受体的哪些区域是配体依赖所必需的内化，然后建立表达a基因的垂体细胞系内化或内化异常的突变受体的种类回收利用。然后我们将测试配体依赖的可能性内化是导致脱敏或 TRH反应的再增敏。我们已经展示了单元格显示一个如果暴露在TRH脉冲下，他们对TRH的钙反应会受到损害间隔很近，但对TRH脉冲的完全响应间隔为每隔至少十分钟。我们将比较细胞对钙离子的反应表达正常或内化缺陷的受体。我们还将测定选择性抑制内化后的TRH反应或者回收利用。最后，我们假设当大多数受体回收时在内化之后，一部分受体被分类到一个降解的，溶酶体途径，说明逐渐同源降低监管。我们将通过测量以下能力来检验这一假设 TRH调节细胞内表达内化的受体密度- 有缺陷的受体，以及在内吞和循环的细胞中被选择性地抑制了。