There no longer appears to be any question that the vitamin D metabolite, 1alpha,25- dihydroxyvitamin D3 (1alpha,25-(OH)2D3, calcitriol or CT), and its metabolic and synthetic analogs manifest nongenomic actions. Rapidly emerging evidence indicates that the nongenomic action plays important roles in controlling the mechanisms that underlie many diseases arising from disturbances in calcium homeostasis (e.g., osteoporosis), cell growth (breast carcinoma, psoriasis), cell differentiation and immune responses (possibly AIDS). The recent advent of noncalcemic, synthetic analogs has ushered in the realistic possibility of therapeutic use of these synthetic agents for the treatment of the above-mentioned diseases. The most prominent evidence for the nongenomic action of 1alpha,25- (OH)2D3 is stimulation of an acute, transient increase in the intracellular free calcium concentration ([Ca2+]I) and concomitant production of inositol polyphosphates. These are the first measurable manifestations that occur within one minute of cellular exposure to the agents. These actions closely resemble those of membrane (cell surface) receptor-agonist mediated mechanisms. However, the precise mechanisms by which vitamin D analogs induce the acute change in [Ca2+]I are unknown. The applicants' recent data demonstrate that the vitamin D receptor (VDR) binds to both plasma membrane (PM) and endoplasmic reticulum (ER). Furthermore, calreticulin (CRT) in the ER fraction is a specific acceptance protein for VDR. Since these two membranes constitute the major sites of [Ca2+]I control, the interaction between VDR and the membranes, and the role of the interaction in [Ca2+]I control must be clarified. Recent advances in cell biology (transfection technology), synthesis of vitamin D analogs with different biological properties and technologies related to the measurement of calcium fluxes in living cells, as well as in isolated membrane vesicles, make it now possible to logically investigate the problems at hand. Therefore, the Specific Aims of the proposed studies are to: 1) analyze the interaction of the VDR with the ER in order to elucidate the role of VDR/CT interaction on Ca2+ release from the ER and to determine the function of CRT in regulating intracellular Ca2+ stores; and 2) study the interaction of the VDR with the PM in order to elucidate the role of this interaction in acute Ca2+ influx stimulated by CT, and to identify a PM-specific acceptance protein for VDR.
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