Modulation of Receptor Number in Cultured Cells

描述（由申请人提供）：下丘脑促甲状腺素释放激素（TRH）刺激垂体前叶释放促甲状腺素和催乳素。TRH受体是一种Ca2+动员G蛋白偶联受体，其失活机制尚不清楚。脱敏是由受体磷酸化、β -抑制蛋白结合和内化引起的。再敏化需要受体去磷酸化和再循环。实际上没有关于TRH受体在体内实际使用情况的信息。我们最近开发了一种针对磷酸化（即最近激活的）TRH受体的高选择性磷酸化位点特异性抗体，使我们能够解决这些问题。该抗体识别的磷酸化位点对受体内化和脱敏至关重要，该抗体对trh注射大鼠的垂体组织具有强染色作用。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，并确定受体激活如何在体内发生。第一个目标是使用新技术来确定磷酸化的化学计量。将确定所涉及的激酶，以及受体上的磷酸化位点，然后将其突变并评估对信号传导，运输和脱敏的影响。下一个目标将测试一个假设，即去磷酸化对控制内化受体的细胞内运输至关重要。将测量质膜和内吞囊泡上受体的去磷酸化速率，并确定去磷酸化的亚细胞位点。第三个目的是验证TRH受体循环和再敏化是由磷酸化依赖性β -抑制蛋白结合控制的假设。确定TRH受体循环的途径。将定义β -抑制素在TRH受体循环和再敏化中的作用，以及受体二聚化的重要性。这些目标中的大多数实验将使用垂体GH3细胞进行。最后一个目的是利用磷酸化位点特异性抗体识别最近激活的TRH受体的能力。该抗体将用于跟踪大鼠和TRH和TRH受体敲除小鼠在服用TRH后的受体磷酸化，并测试下丘脑TRH驱动是体内TSH输出变化的原因，以响应甲状腺状态、营养和温度的变化。

DESCRIPTION (provided by applicant): Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyrotropin and prolactin release by the anterior pituitary gland. 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, trafficking and 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 receptors on the plasma membrane and in endocytic vesicles will be measured, and the subcellular sites of dephosphorylation will be determined. The third aim tests the hypothesis that TRH receptor cycling and resensitization are controlled by phosphorylation-dependent beta-arrestin binding. The pathway of TRH receptor recycling will be determined. The role of beta-arrestin in TRH receptor cycling and resensitization will 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.