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

1,25D3-MARRS 受体激活的信号通路研究

基本信息

批准号：
8098087
负责人：
Susan Ellen Safford
金额：
$ 20.99万
依托单位：
LINCOLN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-29 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8098087
关键词：
Affinity Antibiotics Antibodies Area Binding Binding Proteins Biochemical Pathway Bioinformatics Biology Biotechnology Bone Diseases Cardiovascular Diseases Cardiovascular system Caveolins Cell Line Cell membrane Cell model Cells Cellular Structures Cellular biology Characteristics Chemistry Chickens Collaborations Colon Carcinoma Colorectal Cancer Complement Complex Delaware Department of Defense Development Disease ERp57 Electrophoresis Enterochromaffin Cells Epithelial Epithelial Cells Equipment Eukaryota Exhibits Funding Funding Opportunities G-substrate GTP-Binding Proteins Gas Chromatography Gel Gifts Health Homologous Gene Human Immunoblotting Immunoprecipitation Individual Interphase Cell Intestines Investigation Investigator-Initiated Research Isoelectric Focusing Laboratories Lead Letters Lipofectamine Malignant Neoplasms Malignant neoplasm of prostate Mammalian Cell Mediating Membrane Membrane Proteins Methods Modification Molecular Molecular Biology Nuclear Nuclear Receptors Ohio Osteomalacia Pathway interactions Physiological Plasmids Precipitation Proteins Proteomics Publications Quail Rattus Reader Reporting Research Research Design Research Proposals Rickets Scaffolding Protein Signal Pathway Signal Transduction Signaling Molecule Signaling Protein Site Specificity Spectrophotometry Spottings Steroids Students System Tissues Training Transfection Two-Dimensional Gel Electrophoresis United States National Institutes of Health Universities Utah Visit Vitamin D Vitamin D3 Receptor Western Blotting Work age related cDNA Library caveolin 1 cell type crypt cell data sharing design disorder prevention improved millisecond non-genomic novel overexpression prevent receptor receptor binding response sarcoma scaffold two-dimensional working group

项目摘要

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.

描述（由申请人提供）：维生素D（1，25（OH）2d3）的活性形式与许多疾病状态有关，包括rick骨，骨质乳酸和心血管疾病（Hollick，2004年），以及前列腺，结肠癌，结肠癌和结直肠癌（Deeb等人，2007年）。与健康和疾病相关的1，25（OH）2d3的某些作用是通过核受体引起的；然而，许多作用是膜发射的，1，25（OH）2d3的相互作用可能与新型膜相关的快速反应类固醇（1,2503-MARR）结合蛋白，该蛋白最近由我们的组鉴定出来（Nemere等，2004a）。我们建议1,2503-Marrs是1，25（OH）2d3的膜结合受体，并且至少负责与1，25（OH）2d3相关的膜发射作用。最近在一份评论中，Fleet（2004）指出：“ ...膜发起的信号系统需要更广泛地表征……”。因此，这项研究的具体目的旨在表明1,25d3-marrs结合1，25（OH）2d3发生在质膜上，并负责激活PKCA，并识别一些导致PKCA活化的分子相互作用。研究的分子相互作用将是特定的G蛋白，核维生素D受体以及脚手架蛋白可爱蛋白和RACK-1。这些方法包括使用具有良好特征的，1,2503-Marrs转染的鹌鹑细胞系和两个大鼠肠上皮细胞系，对1,25d3的反应差异。细胞将用1、25（OH）2d3或媒介物处理，分离的质膜，用各种抗体免疫沉淀的蛋白质，并单独通过SDS/PAGE或单独或二维电泳分离，然后进行蛋白质印迹或质量分光光度分析。这些方法将使我能够确定质膜中哪些蛋白质相关，并鉴定免疫沉淀复合物中未知或意外的蛋白质。特定的细胞系应使我能够在1，25（OH）2d3刺激的初始激活途径中确定关键蛋白。在许多研究中已经显示了维生素D，以防止某些癌症及其最初发生，以防止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对细胞的分子作用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。