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

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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.