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

描述(申请人提供)：异源三聚体G蛋白在细胞信号传递中起着重要作用，它将细胞表面的G蛋白偶联受体(GPCRs)接收到的配体结合信息传递到质膜表面的效应酶和离子通道上。由于具有结合G蛋白β/伽马亚单位二聚体的能力，一系列的蛋白质被认为参与了G蛋白信号转导。然而，他们在这一进程中的作用仍然难以捉摸。最近有报道称，光导蛋白家族中的一个成员--光导蛋白样蛋白(PhLP1)是从其新生多肽中组装Gβ/伽马二聚体所必需的。PDC家族的其他成员PhLP2和PhLP3不结合Gβ/伽马，但它们也参与了其他蛋白质折叠过程。鉴于这些新的发现，似乎PhLPs的主要功能可能是帮助折叠新生多肽并将这些蛋白质组装成复合体。这些实验旨在确定PhLP1催化Gβ/伽马组装的分子机制，并阐明PhLP2和PhLP3在蛋白质折叠中的作用。为此，该提案的具体目的是：1)确定依赖磷酸化的PhLP1-Gbeta从CCT释放的分子机制。2)确定PhLP1磷酸化引起的PhLP1-Gbeta-CCT复合体的构象变化。3)确定PhLP1对不同Gβ/γ亚基组合组装的影响。4)研究PhLP3磷酸化对肌动蛋白和微管蛋白折叠的影响。5)测定PhLP2A在14-3-3蛋白和α-微管蛋白折叠中的功能。实现这些目标的方法是多学科的，包括RNA干扰、细胞转染、免疫沉淀、定点突变、蛋白质折叠和亚单位组装分析、冷冻电子显微镜、X射线结晶学和蛋白质及其磷酸化位点的质谱学鉴定。这些拟议的研究一般涉及蛋白质折叠的基本问题，特别是G蛋白β/伽马二聚体的形成。因此，他们将提供重要的信息，以了解与蛋白质错误折叠有关的病理，如阿尔茨海默氏症、普里恩和亨廷顿病，以及在G蛋白通路中开发新的治疗靶点。

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.