Investigation of the Signaling Pathway Activated by 1,25D3-MARRS Receptor

描述（由申请人提供）：维生素D（1，25（OH）2D 3）的活性形式涉及许多疾病状态，包括佝偻病、骨软化和心血管疾病（Hollick，2004），以及前列腺癌、结肠癌和结肠直肠癌（Deeb等人，2007年）。1，25（OH）2D 3在健康和疾病预防方面的一些作用是通过核受体引发的;然而，许多作用是膜引发的，1，25（OH）2D 3可能与我们小组最近鉴定的新型膜相关快速反应类固醇（1，2503-MARRS）结合蛋白相互作用（Nemere et al，2004 a）。我们认为1，2503-MARRS是1，25（OH）2D 3的膜结合受体，并负责至少一些与1，25（OH）2D 3相关的膜启动作用。在最近的一篇评论中，Fleet（2004）指出，“......膜启动的信号系统需要更广泛地表征。".因此，本研究的具体目的旨在表明1，25（OH）2D 3的1，25 D3-MARRS结合发生在质膜上，并负责激活PKC α，并确定导致PKC α激活的一些分子相互作用。研究的分子相互作用将是特定的G蛋白，核维生素D受体，以及支架蛋白小窝蛋白和RACK-1。所述方法包括使用充分表征的、1，2503-MARRS转染的鹌鹑细胞系和两种对1，25 D3具有差异反应的大鼠肠上皮细胞系。将用1，25（OH）2D 3或溶剂处理细胞，分离质膜，用各种抗体免疫沉淀蛋白质，并单独通过SDS/PAGE或二维电泳分离，然后进行Western印迹或质谱分析。这些方法将使我能够确定哪些蛋白质与质膜相关，并识别免疫沉淀复合物中未知或意外的蛋白质。特定的细胞系应该允许我确定关键蛋白质在初始激活途径刺激的1，25（OH）2D 3。许多研究表明，维生素D可以预防某些癌症的传播，并可能预防其初次发生，预防与年龄相关的骨骼疾病，并可能有助于改善心血管健康。提高我们对维生素D对细胞的分子效应的理解将有助于临床医生在维生素D治疗方面做出更明智的治疗决定。

DESCRIPTION (provided by applicant): The active form of vitamin D (1, 25(OH)2D3) has been implicated in a number of disease states including rickets, osteomalacia, and cardiovascular disease (Hollick, 2004), and prostate, colon, and colorectal cancers (Deeb et al., 2007). Some actions of 1, 25(OH)2D3 in relation to health and disease prevention are iniated through the nuclear receptor; however, many actions are membrane-initiated and the interaction of 1, 25(OH)2D3 may be with a novel Membrane Associated Rapid Response Steroid (1,2503-MARRS) binding protein, recently identified by our group (Nemere et al, 2004a). We propose that 1,2503-MARRS is a membrane binding receptor for 1, 25(OH)2D3 and is responsible for at least some of the membrane-initiated actions associated with 1, 25(OH)2D3. Recently in a review, Fleet (2004) stated that, "...the membrane initiated signaling system needs to be more extensively characterized...". Therefore, the specific aims of this study are designed to show that 1,25D3-MARRS binding of 1, 25(OH)2D3 occurs on the plasma membrane and is responsible for activation of PKCa, and to identify some of the molecular interactions that lead to PKCa activation. Molecular interactions investigated will be specific G proteins, the nuclear vitamin D receptor, and the scaffolding proteins caveolin and RACK-1. The methods include using a well characterized, 1,2503-MARRS-transfected quail cell line and two rat intestinal epithelial cell lines with differential responses to 1,25D3. Cells will be treated with 1, 25(OH)2D3 or vehicle, plasma membranes isolated, proteins immunoprecipitated with various antibodies and separated by SDS/PAGE alone or two dimensional electrophoresis followed by Western blot or mass spectrophotometric analysis. These methods will allow me to determine which proteins are associated in the plasma membrane and to identify unknown or unexpected proteins in the immunoprecipitated complexes. The specific cell lines should allow me to determine key proteins in the initial activation pathway stimulated by 1, 25(OH)2D3. Vitamin D has been shown in numerous studies to prevent the spread of some cancers and possibly their initial occurrence, to prevent age-related bone diseases, and possibly contribute to improved cardiovascular health. Improving our understanding of the molecular effects of vitamin D on the cell will help clinicians make more informed treatment decisions with regards to vitamin D therapy.