Molecular Mechanism of Photoreceptor G Protein Signaling

描述(申请人提供)：该研究计划的长期目标是阐明转导蛋白(GT)信号在视杆细胞和视锥细胞中的分子机制。该领域对光转导级联中的转导机制的了解已经达到了显著的水平。我们目前的研究重点是光转导以外的转导蛋白转运和转导蛋白依赖的光感受器反应的调制机制。关于转导蛋白从内段(IS)到外段(OS)的转运，人们知之甚少。非协调119蛋白(UNC119)已成为促进转导蛋白(G？)转运的主要因素。我们建议用小角X射线散射(SAXS)和X射线结晶学方法研究G？t与UNC119之间的输运络合物的结构和性质。基于SAXS的初步分析，我们推测，在UNC119诱导的GT亚基的解离和膜上蛋白质的增溶过程中，UNC119和G？t之间有一个新的界面是必不可少的。通过突变分析，探讨了UNC119增溶GT的界面和提出的模型。GT从IS膜中增溶形成G？t-UNC119络合物是G？-gt；OS运输的第一步，最终货物释放到OS中。一个小的ARF样3GTP酶，ARL3，将作为G？t贩运中的一个假定释放因子进行调查。我们假设G？t是通过ArL3诱导的构象变化从UNC119的络合物中释放出来的，并将对这一机制进行验证。蛋白质运输缺陷是光感受器退化的常见原因。阐明转导蛋白转运的分子细节将有助于理解光感受器外周膜蛋白的转运，并将对视网膜疾病具有重要意义。最后，我们将研究一个新的概念，关于光分散转导在 视杆突触输出的调制。我们推测，G？t通过改变与主要突触分子CaBP4和Ribeye的相互作用和/或通过直接作用于电压门控的钙通道和突触机制来调节杆-杆双极细胞信号转导。转导蛋白对突触调制的分析可能会深刻地影响光感受器中转导蛋白信号的既定范式。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to elucidate the molecular mechanisms of transducin (Gt) signaling in rods and cones. A remarkable level of understanding has been achieved in the field on the mechanisms of transducin in the phototransduction cascade. The current focus of our research is on the mechanisms of transducin trafficking and transducin-dependent modulation of photoreceptor responses beyond phototransduction. Very little is known about transport of transducin from the inner segment (IS) to the outer segment (OS). Uncoordinated 119 protein (UNC119) has emerged as a major factor facilitating the IS->OS transport of transducin-¿ (G¿t). We propose to investigate the structure and properties of the trafficking complex between G¿t and UNC119 by Small Angle X-ray Scattering (SAXS) and X- ray crystallography. Based on the preliminary SAXS analysis, we hypothesize that a novel interface between UNC119 and G¿t is essential to the UNC119-induced dissociation of Gt subunits and protein solubilization from the membrane. The interface and the proposed model of Gt solubilization by UNC119 will be probed with mutational analysis. Solubilization of Gt from IS membranes with the formation of the G¿t-UNC119 complex is the initial step in the IS->OS transport of G¿t, which terminates with the cargo release into the OS. A small ARF-like 3 GTPase, ARL3, will be investigated as a putative release factor in G¿t trafficking. We hypothesize that G¿t is released from the complex with UNC119 by a mechanism involving ARL3-induced conformational changes both within and outside the acyl-binding cavity of UNC119 and will test this mechanism. Defects in protein transport are a common cause of photoreceptor degeneration. Elucidation of molecular details of transducin trafficking will significantly contribute to the understanding of transport of peripheral membrane proteins in photoreceptors and will have important implications for retinal diseases. Finally, we will investigate a novel concept on the role of light-dispersed transducin in modulation of the synaptic output of rods. We hypothesize that G¿t regulates rod-to-rod bipolar cell signaling by altering UNC119 interactions with major synaptic players, CaBP4 and RIBEYE, and/or via direct effects on the voltage-gated Ca2+ channels and synaptic machinery. The proposed analysis of the synaptic modulation by transducin may profoundly impact the established paradigm on transducin signaling in photoreceptors.