SIGNAL TRANSDUCTION IN G-PROTEIN COUPLED RECEPTORS

该提案的重点是G蛋白偶联受体（GPCR）的信号转导的分子机制。 哺乳动物视紫红质是该家族中的原型受体，并且是本文提出的工作的主要焦点。 提出了一个统一的假设，即在所有的GPCR中，信号转导的分子机制（在视紫红质中的光活化后和在GPCR中的配体结合后）是保守和相似的。 在视紫红质中，光诱导的视网膜异构化的第一个结果已经被确定为跨膜结构域中螺旋的移动。 这些运动诱导细胞质面的特定构象变化。 所涉及的三级结构变化先前已由本PI通过生物化学（化学反应性、氨基酸之间的可及性和邻近关系）以及通过自旋标记和EPR光谱进行了研究。 现在将通过核磁共振更精确地研究这些。 将使用溶液NMR（19 F和TROSY）和固态NMR。 NMR实验所需的蛋白质的大规模表达将通过进一步研究HEK 293稳定细胞系和果蝇表达系统来进行。 蛋白质-蛋白质相互作用导致敏化和脱敏是视紫红质信号转导的核心。 利用表面等离子体共振技术，在Biacore仪器上对光激活视紫红质、转导素和视紫红质激酶之间的相互作用进行了详细的动力学研究。 蛋白质相互作用研究的另一个重点是通过共价交联方法确定相互作用位点和氨基酸序列。 将通过质谱方法鉴定相互作用区域。 最后，现在可以通过研究GPCR的两个成员，α 1肾上腺素能受体和β 2肾上腺素能受体中配体结合的螺旋运动来测试信号转导的统一假设。

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.