Vitamin D has diverse and pleiotropic biological effects. Classically it has been considered to be one of the key regulators of calcium and phosphorous homeostasis in the body. More recently, however, non classical roles as an immunomodulator and regulator of cellular proliferation have become more apparent. Associated with these functions are numerous diseases where alterations of the vitamin D pathway have been implicated. Clearly determination of action of vitamin D will allow elucidation of its role in osteogenesis, odontogenesis, and ultimately lead to advances in the treatment of osteoporosis, osteomalacia and other related disease states. The recent cloning of the vitamin D receptor and comparison of its primary sequence with that of other cloned steroid receptors have indicated clearly that the vitamin D receptor is closely related to the receptors of other steroid hormones. The subsequent development of a vitamin D responsive transcription unit in heterologous cells and the use of reverse genetics has allowed an elucidation of the functional domains of the receptor. More structural information was obtained from studies which identified the molecular defects in receptors isolated from patients with type II vitamin D dependent rickets. However, a more comprehensive analysis of receptor structure and a microdissection of functional domains is required and can best be accomplished by a combined biochemical and genetics approach. In order to advance further our understanding of the mechanism of action of vitamin D receptor, we propose to exploit a new novel eucaryotic host system for studies of vitamin D receptor action. This non-mammalian system (Saccharomyces cerevisiae) will allow us to probe 3 areas of vitamin D action: (A) Receptor structure: Genetic screens will be used to determine the critical amino acids required for ligand binding. (B) The mechanism of activation of transcription: utilizing genetic screens to identify factors other than the receptor required for activation of transcription. (C) Studies of the role of phosphorylation on receptor function. Because of the similarity in the mechanisms of all the steroid receptors, any information gained in this study will advance the knowledge in steroid hormone action as a whole. Furthermore, elucidation of the structures required for hormone binding will complement structural analysis of the molecule which together will ultimately lead to a rational drug design program for vitamin D agonists and antagonists.
