STUDIES ON B2-ADRENERGIC RECEPTOR MRNA BINDING PROTEIN

B2-肾上腺素受体mRNA结合蛋白的研究

• 批准号: 6636271
• 负责人: BABY G THOLANIKUNNEL
• 金额: $ 18.33万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636271
• 关键词: G protein; RNA binding protein; RNA biosynthesis; beta adrenergic receptor; cell line; complementary DNA; messenger RNA; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein purification; protein structure function; protein transport; receptor binding; receptor sensitivity

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.

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 蛋白相关受体 (GPLR) 表达的机制，采用 β 肾上腺素能受体 (β2 AR) 作为此类 300 多种受体的原型。我建议探索 GPLR 超家族许多成员共有的一个特征，即激动剂诱导的受体下调。迄今为止，大多数研究仅关注短期脱敏机制。这些机制包括翻译后蛋白修饰（如磷酸化）、与调节蛋白（如视紫红质抑制蛋白和受体激酶）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改变其表达水平。这可能是通过改变蛋白质降解、回收、蛋白质合成的速率，或通过改变编码受体的 mRNA 的合成和降解速率来实现的。我过去六年的工作重点是后者的控制机制。我们已经鉴定并表征了纯化的 35,000 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 β2-受体 mRNA 结合，表现出激动剂诱导的不稳定。通过 UV 交联标记转移鉴定的该蛋白质的表达水平与受体 mRNA 成反比变化。这种蛋白质似乎参与了一种非常新颖的调节途径，因为它似乎会破坏受体 mRNA 的稳定性。我对此提案的具体目标如下： #1) 获得序列信息并产生纯化的 betaARB 蛋白的抗体，并使用这些信息对 betaARB 蛋白的全长 cDNA 进行分子克隆。 #2) 研究 betaAR5B 蛋白参与 beta2AR mRNA 降解的机制并表征该多肽的细胞内特性。