FOLLISTATIN--MOLECULAR STRUCTURE/FUNCTION

尽管卵泡抑素最初是从卵泡液中分离出来的 作为垂体FSH分泌的调节剂，它迅速得到了人们的认可 作为许多组织中细胞发育的重要局部介质 通过其强大的结合激活素的能力。除了生殖 功能，卵泡抑素影响软骨细胞和破骨细胞发育 在软骨内骨形成过程中，并参与造血， 胰岛细胞功能和神经发育。288个残基分子 包括三个独特的10-半胱氨酸结构域，前面有63个残基 N-末端片段，似乎对激活素结合很重要。 值得注意的是，卵泡抑素结构域已经在许多 细胞外基质蛋白，其中一些还与生长因子结合， 对细胞间的相互作用产生不同的影响 差异化。人们对其功能意义知之甚少。 卵泡抑素结构域，也不是激活素结合的全部范围 区域及其对三维结构的依赖 分子。该项目将使用蛋白质化学、免疫学和 确定关键结构元素的分子生物学方法 潜在的卵泡抑素作用。具体目标1将决定 二硫键对齐构筑卵泡抑素的分子结构 使用多肽图和序列分析进行链接。结果将会是 揭示结构域是代表“自主的”折叠单元还是 彼此相交。在目标2下，我们将映射激活素结合 使用合成肽的N-末端结构域中的决定因素， 特异性抗体和反应性氨基酸的化学修饰 侧链。将通过激活素的竞争分析来测试效果 体外前列腺中激活素活性的结合和抑制 癌细胞系。重组全长卵泡抑素及其N-端 结构域将在Aim 3和激活素结合下表达 通过定点突变提炼要求。AIM 4的工作 将通过以下方式确定卵泡抑素结构域的功能作用 缺失突变体、单个结构域的表达和突变 在这些域中的站点。这将澄清是否 卵泡抑素结构域主要是结构元素或直接 与激活素结合的N-末端结构域的参与者。一个 卵泡抑素分子的功能模型应该会出现， 加强未来在理解ITS的结构基础方面的进展 在发现它的许多系统中的操作。

Although follistatin was initially isolated from follicular fluid as a regulator of pituitary FSH secretion, it has rapidly gained recognition as an important local mediator of cell development in many tissues through its potent ability to bind activin. Besides reproductive function, follistatin influences chondrocyte and osteoclast development during endochondral bone formation, and is involved in hematopoeisis, islet cell function, and neural development. The 288-residue molecule includes three distinctive 10-cysteine domains, preceded by a 63-residue N-terminal segment that appears important for activin binding. Significantly, follistatin domains have been found in a number of extracellular matrix proteins, some of which also bind growth factors, that exert diverse effects on cell-cell interactions and differentiation. Little is known regarding the functional significance of the follistatin domains, nor the full extent of the activin-binding region and its dependence on the three-dimensional structure of the molecule. This project will use protein-chemical, immunological and molecular-biological methods to define the critical structural elements underlying follistatin action. Specific Aim 1 will determine the molecular architecture of follistatin by alignment of the disulfide linkages using peptide mapping and sequence analysis. The results will reveal whether the domains represent "autonomous" folding units or are crosslinked to one another. Under Aim 2 we will map the activin-binding determinants in the N-terminal domain using synthetic peptides, site- specific antibodies, and chemical modification of reactive amino-acid side-chains. Effects will be tested by competition assays for activin binding and inhibition of activin activity in an in vitro prostate cancer cell line. Recombinant full-length follistatin and N-terminal domain will be expressed under Aim 3 and the activin binding requirements refined by site-directed mutagenesis. The work of Aim 4 will establish the functional role of the follistatin domains through deletion mutants, expression of an individual domain, and mutagenesis at sites within these domains. This will clarify whether the follistatin domains are primarily structural elements or direct participants with the N-terminal domain in activin binding. A functional model of the follistatin molecule should emerge that will enhance future progress in understanding the structural basis for its actions in the many systems in which it is found.