CELL AND MOLECULAR BIOLOGY OF THE TRH RECEPTOR

TRH 受体的细胞和分子生物学

• 批准号: 2443769
• 负责人: THOMAS K GRAVES
• 金额: $ 7.66万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443769
• 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

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.

下丘脑促甲状腺激素释放肽的垂体受体 促甲状腺激素（TRH）是一种G蛋白偶联的钙动员受体。在 对激动剂结合的反应，TRH-受体复合物经历快速和 广泛的内部化。受体贴附于细胞膜 在较长时间内去除配体后，受体经历 同源下调。我们已经直接证明了受体循环 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 在垂体催乳素细胞中。这种快速而广泛的功能 自行车是完全未知的。我们假设配体依赖性 TRH受体的内吞作用和再循环在TRH 反应;具体来说，我们将测试是否需要内化 对于TRH的脱敏、再敏化或下调， 受体的我们已经证明，具有截短的C- 末端不能内化TRH，但可以增加 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性 内化，然后开发表达a 各种在内化中异常的突变受体，或 回收利用。然后，我们将测试配体依赖性 内化是脱敏或 TRH反应的再敏感化。我们已经证明，细胞显示出 如果暴露于TRH脉冲，钙对TRH的反应就会受损 间隔很近，但对TRH脉冲的完全响应间隔为 间隔至少十分钟我们将比较细胞的钙反应 表达正常或内化缺陷受体。我们还将 在选择性抑制内化后测量TRH反应 或回收利用。最后，我们假设，虽然大多数受体受体拮抗剂 在内化后，一部分受体被分选为 降解，溶酶体途径，占逐渐同源 下调我们将测试这个假设，通过测量的能力， TRH调节表达内化的细胞中的受体密度 缺陷受体，以及在细胞中，内吞作用和再循环， 被选择性抑制。