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

描述（由申请人提供）：我们之前的研究已经确定了两种蛋白质，称为PAIRBP1和PGRMC1，它们由颗粒和黄体细胞表达。我们认为这两种蛋白相互作用形成一种膜复合物，作为孕酮的膜受体（P4）。我们进一步提出P4受体复合物介导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。在这项拨款申请中，我们将提出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生物学行为中的作用。这将通过siRNA和过度表达来减少和增加这两种蛋白质的水平。改变这些蛋白的水平对P4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、大鼠黄体细胞和人颗粒/黄体细胞上进行。确定PAIRBP1在PAIRBP1- pgrmc1复合体中的功能。PAIRBP1通过与PGRMC1相互作用来影响P4的作用，至少有三种可能的机制。这些包括与PGRMC1相互作用以形成P4的“最佳结合袋”；2)将PGRMC1和其他未知蛋白组织成P4信号传递所需的大复合体，3)促进PGRMd定位到质膜。确定PAIRBP1-PGRMC1复合物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的作用。将进行研究以确定这两种蛋白是直接结合还是通过中间蛋白间接结合。将确定PGRMC1中负责形成该复合物的氨基酸序列。还将开发特定的策略来破坏PAIRBP1- PGRMC1复合体。这些策略将基于PGRMC1中参与形成该复合体的氨基酸序列。然后评估破坏PAIRBP1-PGRMC1复合物对P4调节卵巢细胞功能的影响。如果正确，这一概念将改变我们对P4在调节卵巢功能中的作用的认识。它还将为开发新的药物指明道路，这些药物可以选择性地影响PAIRBP1-PGRMC1受体复合物，从而在不改变核P4受体的作用的情况下阻断P4的非基因组作用。这些新的药物制剂在开发避孕药、治疗不孕症和某些形式的癌症方面具有实用价值。

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.