The orphan nuclear receptor, Steroidogenic Factor (SF-1), serves as an essential regulator of endocrine function in both the embryo and the adult. Published studies from several labs, including our own, have demonstrated the essential role of SF-1 in endocrine organogenesis, sex differentiation, and the control of steroidogenesis and peptide hormone gene expression. Yet, we still do not know if SF-1 is activated by a classic ligand-dependent mechanism. As a member of the nuclear receptor superfamily, SF-1 contains a conserved DNA binding domain (DBD) and a putative ligand binding domain (LBD) harboring a C-terminal activating (AF2) domain bearing the signature hexamer domain LxxLL motif of all nuclear receptor (LBD) harboring a C-terminal activating (AF2) domain bearing the signature hexamer domain LxxLL motif of all nuclear receptors. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Our recently published study demonstrate that maximal SF-1 mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser203) located in a major activation domain (AF1) of the protein. In this P01 application our goal is to develop a structural framework for understanding how SF-1 functions in normal endocrine physiology. First, we propose to determine the three dimensional high- resolution crystal structure of the AF1 and LBD of SF-1. These studies will be done in collaboration with Dr. Robert Fletterick. Second, in collaboration with Dr. Voiker Doetsch, we will examine the protein structure of SF-1 before and after phosphorylation of Ser203 residue in the AF1 using nuclear magnetic resonance spectroscopy. Third, we will begin to define the structural interfaces of SF-1: coactivator interactions before and after phosphorylation by biochemical and cellular assays and by use of randomized peptide libraries with Dr. Kip Guy (UCSF). If successful our proposed studies with SF-1 would be the first to obtain a crystal structure for an orphan monomeric receptor, and the first to obtain structural information for both AF1 and AF2 domains. Moreover, SF-1 affords a unique opportunity to address the potential role for phosphorylation in regulating nuclear receptor activity at a structural level. Activation of nuclear receptors by signaling cascades is of widespread interest because such a mechanism could modulate, or even bypass, an obligatory requirement for ligand. In instances of disease, such as hormone-insensitive endocrine tissue cancers (prostate and breast cancers), our research is particularly relevant. Given the prominent role of SF-1 in endocrine physiology, our structural studies may also provide new insights into back mechanisms of hormone signaling.
