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Modulation of Receptor Number in Cultured Cells

培养细胞中受体数量的调节

基本信息

批准号：
8013819
负责人：
PATRICIA M. HINKLE
金额：
$ 35.13万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8013819
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

项目摘要

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)刺激促甲状腺激素和催乳素的释放脑下垂体前叶。钙动员G受体TRH失活机制的研究蛋白偶联受体，目前还不是很清楚。受体磷酸化导致脱敏， β-arrestin结合和内化。再增敏需要受体去磷酸化和再循环。关于TRH受体在体内的实际使用情况，几乎没有任何信息。我们最近做了开发了一种高度选择性的针对磷酸化的磷酸位点特异性抗体(即最近激活)TRH受体，使我们能够解决这些问题。识别的磷酸化位点抗体是受体内化和脱敏的关键，并且抗体对注射TRH大鼠的脑垂体组织。该提案的目标是确定TRH受体的特征磷酸化、去磷酸化、再循环和再敏化，以及建立受体如何激活在活体内发生。第一个目的是使用新可用的技术来确定化学计量比磷酸化。将确定所涉及的激酶，以及受体上的磷酸化位点，然后对其进行突变，并评估其对信令、业务和脱敏的影响。下一个 AIM将检验去磷酸化对控制细胞内流量至关重要的假设内化受体。受体在质膜和胞内的去磷酸化速率将测量囊泡，并确定去磷酸化的亚细胞位置。第三目的验证TRH受体的循环和再增敏受磷酸化调控的假说。从属的解除拘束。TRH受体再循环的途径将被确定。的作用去甲肾上腺素在TRH受体循环和再增敏中的作用以及受体的重要性将被定义二聚化。在这些目标上的大多数实验将使用垂体GH3细胞进行。最后一个目标是利用关于磷酸位点特异性抗体识别最近激活的TRH受体的能力。抗体将用于跟踪TRH在大鼠体内以及TRH和TRH受体中的受体磷酸化基因敲除小鼠，并验证下丘脑TRH驱动导致TSH变化的假设体内输出对甲状腺状态、营养和温度变化的反应。

项目成果

期刊论文数量（39）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

siRNA screen identifies the phosphatase acting on the G protein-coupled thyrotropin-releasing hormone receptor.

siRNA 筛选可鉴定作用于 G 蛋白偶联促甲状腺素释放激素受体的磷酸酶。

DOI：
10.1021/cb3004513
发表时间：
2013
期刊：
ACS chemical biology
影响因子：
4
作者：
Gehret,AustinU;Hinkle,PatriciaM
通讯作者：
Hinkle,PatriciaM

Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells.

蛋白激酶 C 的激活会降低 GH3 垂体细胞的 L 型钙通道活性。

DOI：
10.1152/ajpcell.1992.262.5.c1211
发表时间：
1992
期刊：
The American journal of physiology
影响因子：
0
作者：
Haymes,AA;Kwan,YW;Arena,JP;Kass,RS;Hinkle,PM
通讯作者：
Hinkle,PM

Regulation of endogenous melanocortin-4 receptor expression and signaling by glucocorticoids.

糖皮质激素对内源性黑皮质素 4 受体表达和信号传导的调节。

DOI：
10.1210/en.2006-0984
发表时间：
2006
期刊：
Endocrinology
影响因子：
4.8
作者：
Sebag,JulienA;Hinkle,PatriciaM
通讯作者：
Hinkle,PatriciaM

A novel role for pigment genes in the stress response in rainbow trout (Oncorhynchus mykiss).

DOI：
10.1038/srep28969
发表时间：
2016-07-04
期刊：
Scientific reports
影响因子：
4.6
作者：
Khan UW;Øverli Ø;Hinkle PM;Pasha FA;Johansen IB;Berget I;Silva PI;Kittilsen S;Höglund E;Omholt SW;Våge DI
通讯作者：
Våge DI

Signal transduction and hormone-dependent internalization of the thyrotropin-releasing hormone receptor in cells lacking Gq and G11.

缺乏 Gq 和 G11 的细胞中促甲状腺素释放激素受体的信号转导和激素依赖性内化。