MOLECULAR MECHANISM OF PHOTORECEPTOR G PROTEIN SIGNALING

描述（改编自申请人摘要）：长期目标 一个被提议的项目是研究信号传导的分子机制 通过感光细胞G蛋白，转导素（Gt），在脊椎动物的视觉 转导级联Gt的作用和一般调节过程中 视觉兴奋中的激活和关闭阶段是众所周知的。 然而，我们对Gt的分子细节和机制的理解， 与光激发视紫红质（R*）、cGMP-磷酸二酯酶（PDE）和 G蛋白信号传导的视觉调节剂RGS 9在很大程度上仍然不完整。的 Gt和R* 之间相互作用的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的交联，或 针对R* 的特异性GT-α肽探针，然后鉴定 交联位点探测R*/Gt接口的第二种方法将 涉及嵌合Gs-α/GT-α蛋白产生。之间的竞争 视紫红质和对应于视紫红质的选定区域的合成肽 与Gs-alpha/Gt-alpha嵌合蛋白的结合将揭示 R* 和G t上的点到点相互作用区域。 实验和肽竞争研究将指导定点 R* 的突变。在HEK-293细胞中表达的视紫红质突变体将被 分析与GT-α的相互作用以鉴定关键的R* 残基。 还将分析Gs-alpha/Gt-alpha嵌合体的结合和活化 的R*。本分析的目的是阐明GT-α的作用， 已知参与R* 激活的区域，并鉴定新的GT-α 可能与视紫红质相互作用的位点和残基。详细的地图 Gt上的效应物界面将通过以下的扫描诱变来实现 开关I和GT-α的螺旋结构域。全面分析 效应器接口上的GT将提供有价值的见解的机制， Gt激活PDE和Gt-α螺旋结构域在效应细胞中的作用 调控光感受器GTP酶激活蛋白（GAP），RGS 9，刺激GTP GT-α水解，从而加速视觉上的关闭阶段， 级联。我们对RGS 9对GT-α调节的分析将包括检查 RGS 9的磷酸化和其他潜在的翻译后修饰。 RGS 9与G-beta 5L亚基的结合在细胞凋亡中的功能作用 将探索Gt-alpha GT3活性。总的来说，这些研究将有助于 更好地理解视觉级联中的信号机制， 以及其他G蛋白介导的系统，并将提供信息 与视网膜疾病和一般G蛋白功能疾病相关。

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.