STUDIES ON B2-ADRENERGIC RECEPTOR MRNA BINDING PROTEIN

研究计划的一个主要目标是建立在正常和疾病状态下调节G蛋白连接受体（GPLR）表达的机制，采用β-肾上腺素能受体（β 2 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质修饰如磷酸化，与调节蛋白如抑制蛋白和受体激酶的相互作用，以及受体与其他信号传导组分的物理隔离。尽管对调节短期脱敏的机制有强烈的兴趣，但对受体功能的长期调节知之甚少。一种调节受体的机制是改变其表达水平。这可以通过蛋白质降解、再循环、蛋白质合成速率的变化或通过编码受体的mRNA的降解合成速率的变化来发生。我在过去六年的工作集中在后一种控制机制上。我们已经确定了纯化的35，000 Mr蛋白（β ARB = β AR mRNA结合蛋白）的特征。这种蛋白质由β-肾上腺素能激动剂诱导，并与显示激动剂诱导的不稳定的β 2受体mRNA结合。通过UV交联标记转移鉴定的该蛋白的表达水平与受体mRNA成反比。这种蛋白质似乎参与了一种非常新颖的调节途径，因为它似乎使受体mRNA不稳定。我提出这项建议的具体目标如下： #1）为了获得序列信息并产生抗体， 纯化的betaARB蛋白，并使用这些信息进行分子 克隆β ARB蛋白的全长cDNA。 #2）研究betaAR 5 B蛋白 参与β 2 AR mRNA降解，并表征 该多肽的细胞内性质。

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.