This grant renewal continues the efforts of this laboratory to define at the cellular and molecular levels how the steroid hormone 1alpha,25-dihydroxy-vitamin D3 (1,25D) produces its biological responses. Because of its unusual conformational flexibility 1,25D generates a wide array of molecular shapes that are available for binding to its vitamin D receptor (VDR). We postulate that in a target cell, one unique molecular shape of 1,25D binds to VDR localized in the cell nucleus to modulate gene transcription and that a different unique 1,25D shape binds to an alternative ligand pocket of the VDR associated with plasma membrane caveolae resulting in initiation of rapid response signal transduction pathways. The focus of this proposal is to define the actions of those ligand shapes that interact with the VDR in association with caveolae to produce rapid biological responses of the cell. ? ? AIM (1.0): Determine the biochemical nature of the vitamin D receptor (VDR) associated with the caveolae membrane fraction (CMF), the nature of its association with the membrane and its interaction with caveolin-1. The caveolae VDR will subjected to the following: (s) MALDI-TOF mass spectrometry to gain amino acid sequence information; (b) assessment of possible palmitoylation sites on the VDR; and (c) determination of whether caveolin-1, a membrane structural protein, is crucial for VDR functioning in caveolae using caveolin-1 knock out mice. ? ? AIM (2.0): Define the ligand binding properties of the genomic and proposed alternative pockets of the wild type and selected mutant VDRs with kinetic analyses based on the conformational ensemble model using surface plasmon resonance and correlation with biological (both rapid and genomic) responses. ? ? 1,25D/ VDR rapid responses have been linked to the processes of bone remodeling, insulin secretion and UV ultraviolet (UV) damage in skin keratinocytes. An understanding of how the different molecular shapes of 1,25D interact with different sites on the VDR to initiate different biological responses is fundamental to the design of new drug forms of 1,25D that: (a) allow increases in bone formation that may be therapeutically useful in osteoporosis; (b) modulate insulin secretion as opposed to insulin synthesis; and(c) are anti-apoptotic in human keratinocytes, melanocytes and fibroblasts after ultraviolet (UV)-exposure thereby reducing cancer risk. ? ? ?
