Co-chaperone role of phosducin-like protein in G protein subunit assembly

描述（由申请人提供）：异源三聚体G蛋白通过将细胞表面G蛋白偶联受体（GPCR）接收的配体结合信息穿梭至质膜细胞内表面上的效应酶和离子通道，在细胞信号传导中发挥重要作用。凭借结合G蛋白β/γ亚基二聚体的能力，一段时间以来一直认为蛋白质的Pdc家族参与G蛋白信号传导。然而，它们在这一进程中的作用仍然难以捉摸。最近有报道称，β-葡糖蛋白家族的一个成员，β-葡糖蛋白样蛋白（PhLP 1），是从其新生多肽组装G β/γ二聚体所必需的。Pdc家族的其他成员PhLP 2和PhLP 3不结合G β/γ，但它们也参与其他蛋白质折叠过程。鉴于这些新发现，PhLP的主要功能似乎可能是帮助新生多肽折叠并将这些蛋白质组装成复合物。拟议的实验旨在确定PhLP 1催化G β/γ组装的分子机制，并阐明PhLP 2和PhLP 3在蛋白质折叠中的作用。为此，该提案的具体目的是：1）定义PhLP 1-G β从CCT中磷酸化依赖性释放的分子机制。2)鉴定PhLP 1磷酸化诱导的PhLP 1-Gbeta-CCT复合物的构象变化。3)确定PhLP 1对不同G β/γ亚基组合组装的影响。4)评估PhLP 3磷酸化对肌动蛋白和微管蛋白折叠的影响。5)确定PhLP 2A在14-3-3蛋白和α-微管蛋白折叠中的功能。用于实现这些目标的方法是多学科的，它们包括RNA干扰、细胞转染、免疫沉淀、定点诱变、蛋白质折叠和亚基组装测定、冷冻电子显微镜、X射线晶体学和蛋白质及其磷酸化位点的质谱鉴定。这些拟议的研究解决了蛋白质折叠的基本问题，特别是G蛋白β/γ二聚体的形成。因此，他们将提供重要的信息，在理解与蛋白质错误折叠，如阿尔茨海默氏症，朊病毒和亨廷顿病，以及在G蛋白途径的新的治疗目标的发展病理学。

DESCRIPTION (provided by applicant): Heterotrimeric G proteins play a fundamental role in cell signaling by shuttling the ligand binding information received by G protein-coupled receptors (GPCRs) on the cell surface to effector enzymes and ion channels on the intracellular face of the plasma membrane. By virtue of an ability to bind the G protein beta/gamma subunit dimer, the phosducin (Pdc) family of proteins has been thought for some time to participate in G protein signaling. However, their role in this process has remained elusive. It has recently been reported that a member of the phosducin family, phosducin-like protein (PhLP1), is required for assembly of the G beta/gamma dimer from its nascent polypeptides. Other members of the Pdc family, PhLP2 and PhLP3, do not bind G beta/gamma, but they have also been implicated in other protein folding processes. Given these new findings, it appears that the primary function of PhLPs may be to assist in the folding of nascent polypeptides and the assembly of these proteins into complexes. The proposed experiments seek to determine the molecular mechanism by which PhLP1 catalyzes G beta/gamma assembly and to elucidate the role of PhLP2 and PhLP3 in protein folding. To this end, the specific aims of the proposal are to: 1) Define the molecular mechanism of phosphorylation-dependent release of PhLP1-Gbeta from CCT. 2) Identify conformational changes in the PhLP1-Gbeta-CCT complex induced by PhLP1 phosphorylation. 3) Determine the effects of PhLP1 on the assembly of different Gbeta/gamma subunit combinations. 4) Assess the effects of PhLP3 phosphorylation on actin and tubulin folding. 5) Determine the function of PhLP 2A in 14-3-3 protein and alpha-tubulin folding. The methods used to achieve these aims are multi-disciplinary and they include RNA interference, cell transfection, immunoprecipitation, site-directed mutagenesis, protein folding and subunit assembly assays, cryo-electron microscopy, X-ray crystallography and mass spectrometric identification of proteins and their phosphorylation sites. These proposed studies address fundamental issues in protein folding in general and G protein beta/gamma dimer formation in particular. As such, they will provide information important in understanding the pathologies associated with protein misfolding such as Alzheimer's, prion and Huntington's disease as well as in the development of novel therapeutic targets in G protein pathways.