BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

这项建议的长期目标是阐明人β2-肾上腺素能受体(BetaAR)在天然配体肾上腺素(肾上腺素)和肾上腺素药物类似物刺激下的激活和脱敏机制。肾上腺素对β-AR的刺激参与了许多细胞过程的控制，如肺平滑肌的松弛、心肌收缩的速度和力量、糖原代谢和糖异生的控制等。由于它的许多重要作用，β-AR是许多药物的靶标，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依赖的蛋白激酶和β-AR特异性激酶的磷酸化，一种名为β-arrestin的蛋白质的结合，以及β-AR从质膜进入细胞内部(内吞作用)。这项建议的第一个目标是利用基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱仪，在未经刺激的基础状态下以及在强激动剂(如肾上腺素)和弱激动剂(如沙丁胺醇)模拟后，识别βAR中被磷酸化的氨基酸。将特别强调确定不同激动剂的磷酸化的时间和浓度依赖性。所有研究都将在培养的人类胚胎肾脏细胞中进行，这些细胞要么是野生型BetaAR，要么是特别设计的表位修饰的BetaAR。第二个目的是确定修饰被认为参与定点突变脱敏的β-AR结构域的功能效应。我们将特别关注那些被PKA和BetaAR特异的蛋白激酶磷酸化并影响β-arrestin结合的氨基酸，尽管我们也将研究其他可能参与生长因子调节BetaAR的结构域、BetaAR的棕榈酸化以及与PDZ结构域的结合。第三个目标是通过使用AIMS I和II积累的数据以及从磷酸化/去磷酸化动力学的其他研究中获得的数据，通过数学建模来研究βAR的脱敏、磷酸化、内化和再循环之间的相互关系。

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.