BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

β-肾上腺素能受体结构和脱敏

• 批准号: 6606900
• 负责人: RICHARD B CLARK
• 金额: $ 29.8万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606900
• 关键词: Xenopus oocyte; arrestins; beta adrenergic agent; beta adrenergic receptor; beta adrenergic receptor kinase; cell line; chemical kinetics; chemical models; fatty acylation; insulin; intermolecular interaction; mathematical model; matrix assisted laser desorption ionization; molecular site; palmitates; phosphorylation; posttranslational modifications; protein kinase A; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity; site directed mutagenesis; tyrosine

The long-term objectives of this proposal are to elucidate the mechanisms of activation and desensitization of the human beta2- adrenergic receptor (betaAR) in response to stimulation by the natural ligand epinephrine (adrenalin) and drug analogues of epinephrine. Epinephrine stimulation of the betaAR is involved in the control of many cellular processes such as relaxation of lung smooth muscle, the speed and force of contraction of heart muscle, and the control of glycogen metabolism and gluconeogenesis. Because of its many important roles, the betaAR is the target of many drugs such as albuterol and salmeterol that are mainstays in the treatment of asthma,. The action of epinephrine is rapidly attenuated or desensitized by a complex series of events that serve to shut the receptor down. These processes include phosphorylation by cAMP-dependent protein kinase and betaAR-specific kinases, the binding of a protein called beta-arrestin, and the movement of the betaAR from the plasma membrane into the cell's interior (endocytosis). The first aim of this proposal is to identify the amino acids in the betaAR that are phosphorylated both in the unstimulated, basal state and after simulation by strong agonists such as epinephrine and weak agonists such as albuterol using matrix-assisted laser desorption ionization time- of-flight (MALDI-TOF) mass spectrometry. Particular emphasis will be placed on determining the time and concentration dependency of the phosphorylations by the various agonists. All studies will be perfomed in cultured human embryonic kidney cells that are transfected with either the wild type betaAR or specially engineered epitope-modified betaARs. The second aim is to determine the functional effects of modifying betaAR domains proposed to be involved in desensitization by site- directed mutagenesis. Particular focus will be placed on those amino acids that are phosphorylated by PKA and betaAR-specific protein kinases, and that affect beta-arrestin binding, although we will also examine other domains possibly involved in growth factor regulation of the betaAR, in palmitoylation of the betaAR, and in binding to PDZ domains. The third aim is to examine the interrelationships of desensitization, phosphorylation, internalization and recycling of the betaAR by mathematical modeling using data accumulated from aims I and II as well as from additional studies of phosphorylation/dephosphorylation kinetics.

本提案的长期目标是阐明人β 2-肾上腺素能受体（β AR）对天然配体肾上腺素（肾上腺素）和肾上腺素药物类似物刺激的激活和脱敏机制。肾上腺素对β AR的刺激涉及许多细胞过程的控制，例如肺平滑肌的松弛、心肌收缩的速度和力量以及糖原代谢和糖原生成的控制。由于它的许多重要作用，β AR是许多药物的靶点，如沙丁胺醇和沙美特罗，这些药物是治疗哮喘的主要药物。肾上腺素的作用被一系列复杂的事件迅速减弱或脱敏，这些事件有助于关闭受体。这些过程包括cAMP依赖性蛋白激酶和β AR特异性激酶的磷酸化，称为β-抑制蛋白的蛋白质的结合，以及β AR从质膜进入细胞内部的运动（内吞作用）。该提议的第一个目的是使用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱法鉴定β AR中在未刺激的基础状态下和在通过强激动剂（如肾上腺素）和弱激动剂（如沙丁胺醇）模拟后磷酸化的氨基酸。特别强调的是将被放置在确定由各种激动剂的磷酸化的时间和浓度依赖性。所有研究将在用野生型β AR或特别工程化的表位修饰的β AR转染的培养的人胚肾细胞中进行。第二个目的是确定通过定点诱变修饰被提出参与脱敏的β AR结构域的功能效应。特别的重点将放在那些被PKA和β AR特异性蛋白激酶磷酸化的氨基酸上，这些氨基酸影响β-抑制蛋白结合，尽管我们也将研究可能参与β AR的生长因子调节、β AR的棕榈酰化和与PDZ结构域结合的其他结构域。第三个目标是通过数学建模，使用从目标I和II以及从磷酸化/去磷酸化动力学的额外研究中积累的数据，检查β AR的脱敏、磷酸化、内化和再循环的相互关系。