Molecular Mechanism of Photoreceptor G Protein Signaling

描述（由申请人提供）：本研究计划的长期目标是阐明杆状细胞和视锥细胞中转导蛋白（Gt）信号传导的分子机制。在光导级联中的转导蛋白机制方面已经取得了显著的认识。我们目前的研究重点是转导蛋白的转运机制和转导蛋白依赖的光感受器反应的调节。关于转导蛋白从内段（is）到外段（OS）的转运，我们所知甚少。Uncoordinated 119 protein （UNC119）已成为促进转导蛋白（转导蛋白）在IS->OS转运的主要因子。本文拟利用小角X射线散射（SAXS）和X射线晶体学研究G - t与UNC119之间的交换复合物的结构和性质。基于初步的SAXS分析，我们假设UNC119和Gt之间的新界面对UNC119诱导的Gt亚基解离和蛋白质从膜上溶解至关重要。本文将通过突变分析探讨UNC119对Gt增溶作用的界面和模型。随着G - unc119络合物的形成，Gt从IS膜上的增溶是G ->OS转运的第一步，这一过程随着货物释放到OS而终止。一种小的类似arf的3gtpase， ARL3，将作为假定的G贩运释放因子进行调查。我们假设G通过arl3诱导的UNC119酰基结合腔内外构象变化的机制从UNC119复合物中释放出来，并将对这一机制进行测试。蛋白质运输缺陷是光感受器变性的常见原因。阐明转导蛋白转运的分子细节将有助于理解外周膜蛋白在光感受器中的转运，并对视网膜疾病具有重要意义。最后，我们将探讨一个关于光分散转导蛋白作用的新概念

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.