Modulation of Receptor Number in Cultured Cells

培养细胞中受体数量的调节

• 批准号: 8009683
• 负责人: PATRICIA M. HINKLE
• 金额: $ 5.57万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-19 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009683
• 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.

下丘脑甲状腺激素释放激素（TRH）刺激甲状腺激素和催乳素释放 前垂体。 TRH受体失活的机理，一种Ca2+的g g 蛋白质偶联的受体尚不清楚。受体磷酸化，脱敏的结果， β-arrestin结合和内在化。复敏需要受体去磷酸化和回收利用。 几乎没有关于如何实际在体内使用TRH受体的信息。我们最近有 开发了针对磷酸化的高度选择性的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。由 抗体对于受体内在化和脱敏至关重要，抗体对 来自TRH注射大鼠的垂体组织。该提案的目标是表征TRH受体 磷酸化，去磷酸化，回收和敏化，并建立受体激活如何 发生在体内。第一个目的使用新近可用的技术来确定化学计量法 磷酸化。涉及的激酶将被鉴定，受体上的磷酸化位点也将被鉴定 然后将突变，并评估对信号传导，运输和脱敏的影响。下一个 AIM将检验以下假设：去磷酸化对于控制的细胞内流量至关重要 内部化受体。受体去磷酸化的质膜和内吞作用的速率 将测量囊泡，并确定去磷酸化的亚细胞位点。第三 AIM检验TRH受体循环和脱敏化的假设是由磷酸化控制的 依赖的detaarrestinbinding。将确定TRH受体回收的途径。的作用 在TRH受体循环和脱发化中，将定义Detaarrestin，受体的重要性也将 二聚化。这些目标中的大多数实验都将使用垂体GH3细胞进行。最后的目标资本化 关于磷酸位点特异性抗体鉴定最近活化的TRH受体的能力。抗体 将在大鼠和TRH和TRH受体中的TRH推注后跟随受体磷酸化 敲除小鼠，并检验下丘脑TRH驱动器的假设是TSH的变化 响应甲状腺状态，营养和温度的变化而在体内输出。