SIGNAL TRANSDUCTION IN G-PROTEIN COUPLED RECEPTORS

本文主要研究g蛋白偶联受体（GPCR）信号转导的分子机制。哺乳动物视紫红质是该家族的原型受体，也是本文工作的重点。一个统一的假设是，在所有的GPCR中，信号转导的分子机制（在紫红质中光激活后和在GPCR中配体结合后）都是保守的和相似的。在视紫红质中，光诱导的视网膜异构化的第一个后果已经确定是跨膜结构域中螺旋的运动。这些运动引起细胞质表面的特定构象变化。先前已经通过生物化学（化学反应性，氨基酸之间的接近性和接近关系）以及自旋标记和EPR光谱研究了该PI所涉及的三级结构变化。这些现在将被核磁共振更精确地研究。溶液核磁共振（19F和TROSY）和固态核磁共振都将被使用。通过对HEK293稳定细胞系和果蝇表达系统的进一步研究，将实现核磁共振实验所需蛋白的大规模表达。导致致敏和脱敏的蛋白-蛋白相互作用是视紫红质信号转导的核心。光活化视紫红质、转导蛋白和视紫红质激酶之间相互作用的详细动力学研究将通过使用Biacore仪器的表面等离子体共振技术进行。用共价交联方法确定蛋白质相互作用的位点和氨基酸序列是蛋白质相互作用研究的一个进一步热点。相互作用区域将通过质谱方法确定。最后，通过研究GPCR的两个成员α 1肾上腺素能受体和β 2肾上腺素能受体在配体结合上的螺旋运动，验证信号转导的统一假设是可行的。

This proposal focuses on molecular mechanisms of signal transduction by G-protein coupled receptors (GPCR). Mammalian rhodopsin is the prototypic receptor in this family and is the major focus of the work herein proposed. A unifying hypothesis is proposed that in all GPCR, the molecular mechanisms of signal transduction (after light activation in rhodopsin and after ligand binding in GPCR in general) are conserved and similar. In rhodopsin, the first consequence of light-induced retinal isomerization has been established to be movements of helices in the transmembrane domain. These movements induce a specific conformational change in the cytoplasmic face. The tertiary structure changes involved have previously been studied by this PI by biochemical (chemical reactivity, accessibility and proximity relationships between amino acids) and by spin labeling and EPR spectroscopy. These will now be studied more precisely by NMR. Both, solution NMR (19F and TROSY,) and solid state NMR will be used. Large scale expressions of proteins which are necessary for NMR experiments will be carried out by further investigation of HEK293 stable cell lines and by the Drosophila expression system. Protein-protein interactions leading to sensitization and desensitization are at the heart of signal transduction by rhodopsin. Detailed kinetic studies of interactions between light-activated rhodopsin, transducin and rhodopsin kinase will be carried out by the techniques of surface plasmon resonance using a Biacore instrument. A further focus in studies of protein-protein interactions is to define the interacting sites and amino acid sequences by covalent cross-linking methods. The interacting regions will be identified by mass spectrometric methods. Finally, it is now feasible to test the unifying hypothesis proposed for signal transduction by studying helix movements on ligand binding in two members of GPCR, alpha1 adrenergic receptor and beta2 adrenergic receptor.