STUDIES ON B2-ADRENERGIC RECEPTOR MRNA BINDING PROTEIN

研究提案的一个主要目标是建立G蛋白连接受体(GPLR)在正常和疾病状态下的表达调节机制，使用β-肾上腺素能受体(Beta2 AR)作为这类300多种受体的原型。我建议探索GPLR超家族中许多成员共有的一个特征，即激动剂诱导受体下调。到目前为止，大多数研究都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，与调节蛋白如阻滞素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控知之甚少。调节受体的一种机制是通过改变其表达水平。这可以通过改变蛋白质降解、循环、蛋白质合成的速度，或者通过改变编码受体的mRNAs的合成降解速度来实现。我过去六年的工作主要集中在后一种控制机制上。我们已经鉴定并鉴定了一个纯化的35,000 mR蛋白(betaARB=betaAR mRNA结合蛋白)。这种蛋白是由β-肾上腺素能激动剂诱导的，并与表现激动剂诱导的失稳的β-2受体mRNAs结合。经紫外光交联标记转移鉴定，该蛋白的表达水平与受体基因的表达水平成反比。这种蛋白似乎参与了一种非常新的调节途径，因为它似乎破坏了受体mRNA的稳定。我提出这一建议的具体目的如下：1)获得序列信息并为纯化的BetaARB蛋白提高抗体，并利用这些信息分子克隆BetaARB蛋白的全长cDNA.2)研究β-AR5B蛋白参与β-2AR基因降解的机制，并对该多肽的胞内性质进行表征。

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.