PAIRBP & PGRMC1 act as a membrane receptor complex to mediate P4's ovarian action

描述(申请人提供)：我们之前的研究已经确定了两种蛋白质，称为PAIRBP1和PGRMC1，在颗粒细胞和黄体细胞中表达。我们认为这两种蛋白相互作用形成膜复合体，作为孕酮(P4)的膜受体发挥作用。我们进一步认为，这种P4受体复合体介导了P4的非基因组作用。这些作用包括抑制有丝分裂、维持活力和调节颗粒细胞和黄体细胞的类固醇生成能力。在这项拨款提案中，我们将介绍一系列旨在解决以下三个具体目标的实验：确定PAIRBP1和PGRMC1在调节P4‘S生物学行为中的作用。这将通过siRNA和过度表达来耗尽和增加这两种蛋白质的水平。这些蛋白水平的变化对P4‘S结合~3H-P4、抑制有丝分裂和凋亡以及调节类固醇分泌的能力的影响将被监测。这些研究将在大鼠颗粒细胞、大鼠黄体和人颗粒/黄体细胞上进行。确定PAIRBP1-PGRMC1复合体中PAIRBP1的功能。PAIRBP1与PGRMC1相互作用影响P4‘S作用的可能机制至少有三种。这些包括与PGRMC1相互作用：1)形成P4的“最佳结合口袋”；2)将PGRMC1和其他未知蛋白质组织成P4信号所需的大型复合体；3)促进PGRMd定位于质膜。为了确定PAIRBP1-PGRMC1复合体形成所需的分子位点，以及是否需要PAIRBP1-PGRMC1相互作用来介导P4‘S作用。将进行研究，以确定这两种蛋白质是直接结合还是通过中间蛋白质间接结合。将确定PGRMC1中负责形成该复合体的氨基酸序列。还将制定具体的策略来破坏PAIRBP1-PGRMC1复合体。这些策略将基于PGRMC1内参与形成这种复合体的氨基酸序列。然后将评估破坏PAIRBP1-PGRMC1复合体对P4‘S调节卵巢细胞功能能力的影响。如果这一概念正确，将改变我们对P4‘S在卵巢功能调节中的作用的理解。这也将为开发新的药理药物指明方向，这些药物可以选择性地影响PAIRBP1-PGRMC1受体复合体，从而在不改变核P4受体作用的情况下阻断P4‘S的非基因组作用。这些新的药理制剂可能会在开发避孕药以及治疗不孕不育和某些形式的癌症方面发挥作用。

DESCRIPTION (provided by applicant): 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.