MOLECULAR MECHANISM OF PHOTORECEPTOR G PROTEIN SIGNALING

描述(改编自申请人的摘要)： 拟议的计划是研究潜在的信号转导的分子机制 通过脊椎动物视觉中的光感受器G蛋白转导蛋白(GT) 转导级联。GT的作用及一般调控 视觉兴奋中的激活和关闭阶段是很好理解的。 然而，我们对GT的分子细节和机制的了解 与光激视紫红质(R*)、cGMP-磷酸二酯酶(PDE)和 G蛋白信号的视觉调节因子RGS9在很大程度上仍然不完整。这个 GT和R*之间的交互界面将使用照片进行检查 GT-α突变体与单个反应性半胱氨酸残基或 针对R*的特异性GT-α多肽探针，随后鉴定 交叉链接的站点。探测R*/GT接口的第二种方法将 包括产生嵌合的Gs-α/GT-α蛋白。两国之间的竞争 视紫红质和与视紫红质特定区域相对应的合成肽 与Gs-α/GT-α嵌合蛋白的结合将揭示 R*和GT上的点对点相互作用区。交联化的结果 实验和多肽竞争研究将指导现场定向 R*的诱变作用。在HEK-293细胞中表达的视紫红质突变体 分析与GT-α的相互作用，以确定关键的R*残基。 还将分析GS-α/GT-α嵌合体的结合和激活 由R*。这项分析的目标是阐明GT-α的作用 已知的参与R*激活的区域和识别新的GT-α 位点(S)和可能与视紫红质相互作用的残基。详细的地图绘制 GT上的效应器接口将通过扫描突变完成 开关I和GT-α的螺旋结构域。综合分析了 GT上的效应器界面将为研究GT的作用机制提供有价值的见解 GT激活PDE及GT-α螺旋结构域在效应器中的作用 监管。光感受器GTP酶激活蛋白(GAP)RGS9刺激GTP GT-α的水解从而加速了视觉中的关闭阶段 卡斯卡德。我们对RGS9对GT-α调节的分析将包括检查 RGS9的磷酸化和其他潜在的翻译后修饰。 RGS9与G-β5L亚基结合在细胞周期调控中的作用 GT-αGTP酶活性将被研究。总体而言，这些研究将有助于 更好地理解可视化级联AS中的信号机制 以及其他G蛋白介导的系统，并将提供信息 一般与视网膜疾病和G蛋白功能疾病有关。

DESCRIPTION (Adapted from applicant's abstract): The long-term objective of the proposed program is to investigate molecular mechanisms underlying signaling via the photoreceptor G protein, transducin (Gt), in the vertebrate visual transduction cascade. The role and general regulation of Gt during the activation and turnoff phases in visual excitation are well understood. However, our understanding of the molecular details and mechanisms of Gt interactions with photoexcited rhodopsin (R*), cGMP-phosphodiesterase (PDE) and visual regulator of G protein signaling, RGS9, remains largely incomplete. The interface of interaction between Gt and R* will be examined using photo crosslinking of Gt-alpha mutants with a single reactive cysteine residue or specific Gt-alpha peptide probes to R* followed by identification of the crosslinked sites. A second approach to probing the R*/Gt interface will involve generation of chimeric Gs-alpha/Gt-alpha proteins. Competition between rhodopsin and synthetic peptides corresponding to selected regions of rhodopsin for binding to Gs-alpha/Gt-alpha chimeric proteins will reveal the point-to-point interaction regions on R* and Gt. Results of the crosslinking experiments and peptide competition studies will guide the site-directed mutagenesis of R*. Rhodopsin mutants expressed in HEK-293 cells will be analyzed for interaction with Gt-alpha to identify critical R* residues. Gs-alpha/Gt-alpha chimeras will also be analyzed for binding to and activation by R*. The goals of this analysis are to both elucidate the role of Gt-alpha regions known to be involved in activation by R* and to identify novel Gt-alpha site(s) and residues that may interact with rhodopsin. The detailed mapping of the effector interface on Gt will be accomplished by scanning mutagenesis of the switch I and the helical domain of Gt-alpha. Comprehensive analysis of the effector interface on Gt will provide valuable insights into the mechanism of PDE activation by Gt and the role of the Gt-alpha helical domain in effector regulation. Photoreceptor GTPase activating protein (GAP), RGS9, stimulates GTP hydrolysis of Gt-alpha thus accelerating the turnoff phase in the visual cascade. Our analysis of Gt-alpha regulation by RGS9 will include examination of phosphorylation and other potential posttranslational modifications of RGS9. The functional role of the association of RGS9 with the G-beta5L subunit for Gt-alpha GTPase activity will be explored. Overall, these studies will help to achieve a better understanding of signaling mechanisms in the visual cascade as well as in other G protein mediated systems and will provide information relevant to retinal diseases and diseases of G protein function in general.