Our previous studies have identified two proteins, referred to as PAIRBP1 and PGRMC1, that are expressed by granulosa and luteal cells. We propose that these two proteins interact to form a membrane complex that functions as a membrane receptor for progesterone (P4). We further propose that this P4 receptor complex mediates the non-genomic actions of P4. These actions include inhibiting mitosis, maintaining viability and regulating the steroidogenic capacity of granulosa and luteal cells. In this grant proposal we will present a series of experiments designed to address the following three specific aims: To determine the role of PAIRBP1 and PGRMC1 in regulating P4's biological actions. This will be done using siRNA and over expression to deplete and increase levels of these two proteins. The effect of changing the levels of these proteins on P4's ability to bind 3H-P4, inhibit mitosis and apoptosis and modulate steroid secretion will be monitored. These studies will be conducted on rat granulosa cells, rat luteal and human granulosa/luteal cells. To determine the function of the PAIRBP1 in the PAIRBP1-PGRMC1 complex. There are at least three possible mechanisms through which PAIRBP1 can interact with PGRMC1 to influence P4's actions. These include interacting with PGRMC1 to 1) form an "optimal binding pocket" for P4;2) organize PGRMC1 and other unknown proteins into a large complex that is required for P4 signaling and 3) promote PGRMd's localization to the plasma membrane. To determine the molecular site required for the formation of the PAIRBP1-PGRMC1 complex and whether PAIRBP1-PGRMC1 interaction is required to mediate P4's action. Studies will be conducted to determine whether these two proteins bind to each other directly or indirectly via an intermediary protein. The amino acid sequence in PGRMC1 that is responsible for the formation of this complex will be identified. Specific strategies will also be developed to disrupt the PAIRBP1- PGRMC1 complex. These strategies will be based on the amino acid sequence within PGRMC1 that is involved in forming this complex. Then the effect of disrupting the PAIRBP1-PGRMC1 complex on P4's ability to regulate ovarian cell function will be assessed. If correct, this concept will change our understanding of P4's role in regulating ovarian function. It will also point the way toward the development on novel pharmacological agents that could selectively influence the PAIRBP1-PGRMC1 receptor complex and thereby block P4's non-genomic actions without altering the actions of the nuclear P4 receptor. These new pharmacological agents could have utility in the development contraceptives and in treating infertility and some forms of cancer.