Progesterone has a major role in the development, differentiation and function of female reproductive tissues required for pregnancy, and recent clinical data indicate that progesterone increases breast cancer risk and may have a role in established breast cancer. The biological effects of progesterone are mediated by a progesterone receptor (PR) which is a member of the intracellular receptor superfamily of transcription factors; thus the major action of progesterone is thought to be a direct regulation of gene expression. The goal of the previously funded project was to define the molecular basis for how synthetic ligands, used to target and modulate PR activity, exhibit varying agonist and antagonist activities in a tissue/cell-type and gene specific manner. This is a relevant question with implications for development of improved selective ligand modulators of PR. Because the N terminal (AF-1) domain of PR is required for agonist activity of the partial antagonist RU486, as well as cell-type specific activities of PR, we sought to identify novel PR interacting proteins that mediate the activity of the N-terminal domain and consequently the activity of partial antagonists. We identified jun dimerization protein-2 (JDP-2) as such a factor and showed that it mediates AF-1 activity by a novel mechanism involving a cooperative interaction between the DNA binding domain and AF-1 of the receptor. We also unexpectedly identified a motif in the N-terminal domain of PR that interacts with SH3 domains of signaling molecules, most predominantly Src tyrosine kinases. This has led to the discovery that PR has another function as a rapid progesterone-dependent activator of Src/MAP-kinase signaling cascades through activation of Src by a specific SH3 domain displacement mechanism. This finding reveals a new paradigm for PR action independent of gene transcription. The present proposal seeks to continue studies on the role and mechanism of these two fundamentally different actions of PR with the following specific aims. AIM#1 will determine the role of JDP-2 in PR-mediated gene transcription and regulation of selective ligand modulators of PR, and will define the mechanism of JDP-2 as a mediator of the AF-1 activity. AIM#2 will further dissect PR activated cytoplasmic signaling pathways and by use of point mutations that dissociate cell signaling and transcriptional functions of PR, will evaluate the relative contribution of these two actions of PR to biological responses to progesterone in vitro and in vivo. These studies are anticipated to give new insights into the mechanism of action and role of progesterone in normal tissues and in breast cancer.