Physiology & Action of a New Follistatin-Related Protein

卵泡抑素相关蛋白（follistatin -related protein， FSRP）是新近发现的一种糖蛋白，其基因结构和激活素结合特性与激活素结合蛋白follistatin （FS）高度同源。FSRP最初在b细胞白血病中被克隆为一种过表达蛋白，并在许多癌细胞系中表达，这表明它可能与多种肿瘤有关，或者是一种有用的标记物。然而，与FS不同的是，FSRP在胎盘和睾丸中表达最高（FS在卵巢和肾脏中表达最高），表明FSRP在这些组织中具有独特的功能。免疫细胞化学研究表明，FSRP在所有细胞系和原代细胞的细胞核中高度集中，但仅在FSRP表达水平最高的细胞中分泌。这些观察结果表明，FSRP细胞内运输的调控是复杂和独特的，而且，FSRP可能具有核活动，不同于严格的非核FS。转基因小鼠中FSRP的过度表达会破坏卵泡发育，导致雌性不育。因此，本研究的主要目标是确定FSRP在正常和病理生理环境下的生物学功能，并阐明该蛋白的生物化学特征，这些特征决定了其独特的生物学和分布。转基因和基因敲除小鼠，以及几种体外生物测定将用于鉴定FSRP的正常和病理生理作用（Specific Aim 1）。细胞内运输、生物合成调控和FSRP的核功能将在HeLa和人颗粒细胞中使用脉冲追踪标记、免疫沉淀和亲和层析（Specific Aim 2）进行检测。结合亲和力和配体特异性，以及FSRP负责这些活动的功能域和FSRP的核定位将使用位点定向诱变和与FS的结构域交换来检查（Specific Aim 3）。拟议的研究计划的结果将定义FSRP在正常生理中的作用，确定FSRP过表达破坏卵泡发生的机制，并定义导致核定位和分泌蛋白活性的新调节机制。

Follistatin-related protein (FSRP) is a recently discovered glycoprotein whose gene structure and activin binding properties are highly homologous to the activin binding protein follistatin (FS). FSRP was originally cloned as an overexpressed protein in a B-cell leukemia and is expressed in numerous cancer cell lines, suggesting it may be involved with, or be a useful marker for a wide array of tumors. Unlike FS, however, FSRP is most highly expressed in the placenta and testis (FS is highest in the ovary and kidney), indicating that FSRP has unique functions in these tissues. Immunocytochemical studies have demonstrated that FSRP is highly concentrated in the nucleus of all cell lines and primary cells tested, but is secreted only by cells with the highest FSRP expression levels. These observations suggest that regulation of FSRP intracellular trafficking is complex and unique, and further, that FSRP may have nuclear activities distinct from strictly non-nuclear FS. Overexpression of FSRP in transgenic mice disrupts follicular development, resulting in female infertility. Thus, the broad goal of this proposal is to determine the biological functions of FSRP in normal and pathophysiological circumstances, as well as to elucidate the biochemical features of this protein which govern its unique biology and distribution. Transgenic and knockout mice, along with several in vitro bioassays will be utilized to identify the normal and pathophysiological actions of FSRP (Specific Aim 1). Intracellular trafficking, regulation of biosynthesis, and nuclear functions of FSRP will be examined in HeLa and human granulosa cells using pulse chase labeling, immunoprecipitation, and affinity chromatography (Specific Aim 2). The binding affinity and ligand specificity, as well as functional domains of FSRP responsible for these activities and FSRP's nuclear localization will be examined using site directed mutagenesis and domain swapping with FS (Specific Aim 3). The results of the proposed research program will define the role(s) of FSRP in normal physiology, determine the mechanism whereby FSRP overexpression disrupts folliculogenesis, and define the novel regulatory mechanisms that results in nuclear localization and activity of a protein that is also secreted.