Physiological role of phosducins in the retina

描述（由申请人提供）：真核细胞依赖于G蛋白介导的信号传导途径来检测和响应多种细胞外刺激。基于它们的G蛋白β/γ亚基结合特性，Pdc和Pducin样蛋白（PhLP）被认为调节G蛋白信号传导。然而，最近的证据也表明了其他作用。本研究的长期目标是确定Pdc和PhLP在视网膜感光细胞和其他细胞类型中的生理功能。 最近的研究表明，光调节的光感受器细胞中的Pdc的核定位，这表明Pdc在细胞核中的作用。在特定目的1-3中，研究了调节核定位的潜在机制，并检查了潜在的核结合配偶体。在目的4中，Pdc在光受体中G蛋白的光依赖性易位中的可能作用被解决。在目的5中，研究了PhLP在调节蛋白酶体介导的由细胞外信号调节激酶（ERK）激活引发的G β/γ降解中的作用。 实现这些具体目标的方法是多学科的。它们包括：利用磷酸化位点特异性抗体在培养细胞、视网膜组织和转基因小鼠中进行免疫定位研究;鉴定核结合伙伴的功能蛋白质组学技术;探索泛素/蛋白酶体靶向降解的免疫印迹分析; siRNA介导的基因沉默;鉴定引发降解的磷酸化位点的定点诱变;以及设计用于评估ERK介导的G β/γ降解对G蛋白信号传导的影响的信号传导途径串扰测定。 对Pdc和PhLP的功能的基本理解一直是非常难以捉摸的。本文提出的实验将给出有关这些重要蛋白质在细胞信号传导过程中的作用的生理学相关答案。

DESCRIPTION (provided by applicant): Eukaryotic cells rely on G protein-mediated signaling pathways to detect and respond to a wide variety of extracellular stimuli. Based on their G protein beta/gamma subunit binding properties, phosducin (Pdc) and phosducin-like protein (PhLP) are believed to regulate G protein signaling. However, recent evidence suggests other roles as well. The long term objective of this research is to define the physiological function of Pdc and PhLP in the photoreceptor cells of the retina and other cell types. Recent studies have demonstrated light-regulated nuclear localization of Pdc in photoreceptor cells, suggesting a role for Pdc in the nucleus. In Specific Aims 1-3, potential mechanisms of regulation of nuclear localization are investigated and potential nuclear binding partners are examined. In Aim 4, the possible role of Pdc in light dependent translocation of G proteins in the photoreceptor is addressed. In Aim 5, the role of PhLP in regulating proteasome-mediated degradation of Gbeta/gamma initiated by extracellular signal-regulated kinase (ERK) activation is investigated. The methods used to achieve these specific aims are multi-disciplinary. They include immunolocalization studies in cultured cells, retinal tissues and transgenic mice using phosphorylation site-specific antibodies; functional proteomics techniques to identify nuclear binding partners; immunoblot-based assays to explore ubiquitin/proteasome-targeted degradation; siRNA-mediated gene silencing; site-directed mutagenesis to identify phosphorylation sites that trigger degradation; and signaling pathway crosstalk assays designed to assess the effect of ERK-mediated degradation of Gbeta/gamma on G protein signaling. A basic understanding of the function of Pdc and PhLP has been very elusive. The experiments proposed herein will give physiologically relevant answers concerning the role of these important proteins in cellular signaling processes.