BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

本研究的长期目标是阐明人β 2-肾上腺素能受体（β - aar）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。肾上腺素刺激β - aar参与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，以及糖原代谢和糖异生的控制。由于它的许多重要作用，β - aar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。由于一系列复杂的事件导致受体关闭，肾上腺素的作用迅速减弱或脱敏。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、β -抑制蛋白的结合以及β - aar从质膜进入细胞内部的运动（内吞作用）。本研究的第一个目的是利用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱法，确定β - aar中在未受刺激的基础状态和在强激动剂（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。特别强调将放在确定的时间和浓度依赖于磷酸化的各种激动剂。所有的研究都将在培养的人胚胎肾细胞中进行，这些细胞转染了野生型β - aar或经过特殊工程修饰的β - aar。第二个目的是确定通过位点定向诱变修饰β - aar结构域所涉及的脱敏功能的影响。我们将特别关注那些被PKA和β -ar特异性蛋白激酶磷酸化并影响β -抑制蛋白结合的氨基酸，尽管我们也将研究可能涉及β - aar的生长因子调节、β - aar的棕榈酰化以及与PDZ结构域结合的其他结构域。第三个目标是通过数学建模，利用目标I和II以及磷酸化/去磷酸化动力学的额外研究积累的数据，研究β - aar的脱敏、磷酸化、内化和再循环的相互关系。

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.