Modulation of Receptor Number in Cultured Cells

培养细胞中受体数量的调节

• 批准号: 7545830
• 负责人: PATRICIA M. HINKLE
• 金额: $ 35.84万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545830
• 关键词: Acute; Address; Animals; Anterior Pituitary Gland; Antibodies; Arrestins; Binding; Biological Assay; Bolus Infusion; Cell membrane; Cells; Cultured Cells; Dimerization; Dissociation; Endocytic Vesicle; Endocytosis; Endosomes; Enzyme-Linked Immunosorbent Assay; G-Protein-Coupled Receptors; Goals; Hormones; Hypothalamic structure; Knockout Mice; Label; Learning; Measures; Mole the mammal; Mutate; Organelles; Output; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiologic pulse; Physiological; Pituitary Gland; Prolactin; Protein Dephosphorylation; Rattus; Receptor Activation; Receptor Signaling; Recycling; Role; Signal Transduction; Signal Transduction Pathway; Site; Staining method; Stains; Surface; Techniques; Temperature; Testing; Thyroid Gland; Thyrotropin; Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone Receptors; Tissues; arrestin3; beta-arrestin; desensitization; immunocytochemistry; in vivo; inorganic phosphate; mutant; nutrition; receptor; receptor internalization; receptor recycling; research study; response; stoichiometry; trafficking

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyrotropin and prolactin release by the anterior pituitary gland. The the mechanisms of inactivation of the TRH receptor, a Ca2+-mobilizing G protein-coupled receptor, are not well understood. Desensitization results from receptor phosphorylation, beta-arrestin binding, and internalization. Resensitization requires receptor dephosphorylation and recycling. There is virtually no information about how the TRH receptor is actually used in vivo. We have recently developed a highly selective phospho-site specific antibody against the phosphorylated (i.e. recently activated) TRH receptor that allows us to address these issues. The phosphorylation sites recognized by the antibody are critical for receptor internalization and desensitization, and the antibody gives strong staining of pituitary tissue from TRH-injected rats. The goals of the proposal are to characterize TRH receptor phosphorylation, dephosphorylation, recycling and resensitization, and to establish how receptor activation occurs in vivo. The first aim uses newly available techniques to determine the stoichiometry of phosphorylation. The kinases involved will be identified, as will the phosphorylation sites on the receptor, which will then be mutated and the effects on signaling, traffickingand desensitization assessed. The next aim will test the hypothesis that dephosphorylation is critical for controlling the intracellular traffic of the internalized receptor. Rates of dephosphorylation of receptorson the plasma membrane and in endocytic vesicles will be measured, and the subcellular sites of dephosphosphorylation will be determined. The third aim tests the hypothesis that TRH receptor cycling and resensitization are controlled byphosphorylation- dependent Detaarrestinbinding. The pathway of TRH receptor recycling will be determined. The role of Detaarrestin in TRH receptor cycling and resensitizationwill be defined, as will the importance of receptor dimerization. Most experiments in these aims will be done using pituitary GH3 cells. The last aim capitalizes on the ability of the phospho-site specific antibody to identify recently activated TRH receptor. The antibody will be used to follow receptor phosphorylation following a bolus of TRH in rats and in TRH and TRH receptor knockout mice, and to test the hypothesis that hypothalamic TRH drive is responsible for changes in TSH output in vivo in response to changes in thyroid status, nutrition and temperature.

下丘脑促甲状腺激素释放激素（TRH）通过促甲状腺激素和催乳素的释放， 垂体前叶TRH受体是一种钙离子动员的G 蛋白偶联受体，还没有很好的理解。脱敏是由受体磷酸化引起的， β-抑制蛋白结合和内化。再致敏需要受体去磷酸化和再循环。 事实上，没有关于TRH受体在体内如何实际使用的信息。我们最近 开发了针对磷酸化的高选择性磷酸位点特异性抗体（即最近 激活的）TRH受体，使我们能够解决这些问题。磷酸化位点识别的 抗体对受体内化和脱敏至关重要，抗体对 注射TRH大鼠的垂体组织。该提案的目标是表征TRH受体 磷酸化，去磷酸化，再循环和再敏化，并建立受体活化 发生在体内。第一个目标是使用新的可用技术来确定的化学计量的 磷酸化将鉴定所涉及的激酶，以及受体上的磷酸化位点， 然后将其突变，并评估其对信号传导、贩运和脱敏的影响。下一 aim将检验去磷酸化对于控制细胞内运输的关键假设。 内化受体细胞膜和内吞细胞膜上受体的去磷酸化速率 测量囊泡，并确定脱磷酸化的亚细胞位点。第三 目的是检验TRH受体循环和再敏由磷酸化控制的假设- dependent Detarrestinbinding.将确定TRH受体再循环的途径。的作用 将定义TRH受体循环和再敏中的Detarrestin，以及受体的重要性。 二聚化。这些目的中的大多数实验将使用垂体GH 3细胞进行。最后一个目标是资本化 磷酸化位点特异性抗体识别最近激活的TRH受体的能力。述抗体 将用于跟踪大鼠中TRH推注后的受体磷酸化以及TRH和TRH受体 基因敲除小鼠，并测试假设，下丘脑TRH驱动是负责TSH的变化， 在体内输出响应甲状腺状态，营养和温度的变化。