STUDIES ON B2-ADRENERGIC RECEPTOR MRNA BINDING PROTEIN

研究计划的一个主要目标是建立g蛋白连接受体（GPLRs）在正常和疾病状态下的表达调控机制，以β -肾上腺素能受体（β 2 AR）为原型，对这类300多种受体进行研究。我建议探索GPLR超家族许多成员的共同特征，即激动剂诱导的受体下调。迄今为止，大多数研究只关注脱敏的短期机制。这些机制包括翻译后蛋白修饰，如磷酸化，与调控蛋白（如阻滞蛋白和受体激酶）的相互作用，以及受体与其他信号成分的物理隔离。尽管人们对调节短期脱敏的机制非常感兴趣，但对受体功能的长期调节知之甚少。调节受体的一种机制是通过改变其表达水平。这可能通过蛋白质降解、再循环、蛋白质合成速率的变化，或通过编码受体的mrna降解合成速率的变化而发生。我过去六年的工作重点是后一种控制机制。我们鉴定并鉴定了纯化的35000 Mr蛋白（betaARB=betaAR mrna结合蛋白）。该蛋白由β -肾上腺素能激动剂诱导，并与β - 2受体mrna结合，表现出激动剂诱导的不稳定。该蛋白的表达水平，通过uv交联标签转移鉴定，与受体mRNA呈负相关。这种蛋白似乎参与了一种非常新颖的调节途径，因为它似乎破坏了受体mRNA的稳定。我的具体目的是：#1)获得纯化的betaARB蛋白的序列信息和抗体，并利用这些信息进行betaARB蛋白全长cDNA的分子克隆。#2)研究β aar5b蛋白参与β aar2ar mRNA降解的机制，并表征该多肽的细胞内特性。

A major goal of research proposal is to establish the mechanism by which the expression of G-protein-linked receptors (GPLRs) are regulated in normal and disease states, employing the beta-adrenergic receptor (beta2 AR) as the prototype for this class of more than 300 receptors. I propose to explore a feature common to many members of the superfamily of GPLR, i.e., agonist-induced down-regulation of receptors. Most studies to date have focused only on short-term mechanisms of desensitization. Those mechanisms include post-translational protein modification such as phosphorylation, interactions with regulatory proteins such as arrestins and receptors kinases, and physical sequestration of the receptor away from other signaling components Despite intense interest in the mechanisms which regulate short-term desensitization, very little is known about the long term regulation of receptor function. One mechanism through which a receptor can be regulated by is by altering its level of expression. This could occur through changes in the rate of protein degradation, recycling, protein synthesis, or through changes in the rate of synthesis of degradation of mRNAs encoding the receptors. My work over the last six years as focused on the latter control mechanism. We have identified, characterized a purified a 35,000 Mr protein (betaARB=betaAR mRNA-binding protein). This protein is induced by beta-adrenergic agonists and binds to beta2-receptor mRNAs that display agonist-induced destabilization. The expression level of this protein, identified by UV-cross-linked label transfer varies inversely with receptor mRNA. This protein appears to be involved in a very novel regulatory pathway in that it appears to destabilize the receptor mRNA. My specific aims for this proposal are as follows: #1) To obtain sequence information and raise antibody for the purified betaARB protein and use these information to molecular clone the full length cDNA for betaARB protein. #2) To study the mechanisms by which betaAR5B protein participates in beta2AR mRNA degradation and characterize the intracellular properties of this polypeptide.