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

描述(申请人提供)：维生素D(1，25(OH)2D3)的活性形式与许多疾病状态有关，包括软骨病、骨软化症和心血管疾病(Hollick，2004)，以及前列腺癌、结肠癌和结直肠癌(Deeb等人，2007)。1，25(OH)2D3在健康和疾病预防方面的一些作用是通过核受体启动的；然而，许多作用是膜启动的，1，25(OH)2D3可能与我们小组最近发现的一种新的膜相关快速反应类固醇(1,2503-MARRS)结合蛋白(Nemere等，2004a)。我们认为1,250，3-MARRS是1，25(OH)2D3的膜结合受体，至少参与了与1，25(OH)2D3相关的部分膜启动作用。最近，Fleet(2004)在一篇综述中指出，“……膜启动的信号系统需要更广泛地表征……”。因此，本研究的目的是证明1，25(OH)2D3的1，25D3-MARRS结合发生在质膜上，并负责PKCA的激活，并确定导致PKCA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。这些方法包括使用一个特性良好的、1,2503-MARRS转基因的鹌鹑细胞系和两个对1，25D3有不同反应的大鼠肠道上皮细胞系。将细胞用1，25(OH)2D3或溶剂处理，分离质膜，用各种抗体免疫沉淀蛋白质，然后单独用SDS/PAGE或双向电泳法分离，然后进行Western印迹或质量分光光度分析。这些方法将使我能够确定哪些蛋白质与质膜相关，并在免疫沉淀复合体中识别未知或意想不到的蛋白质。特定的细胞系应该可以让我确定1，25(OH)2D3刺激的初始激活途径中的关键蛋白质。许多研究表明，维生素D可以防止某些癌症的扩散和可能的初始发生，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对细胞分子作用的了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.