STRUCTURE AND FUNCTION OF ANDROGEN-BINDING PROTEIN

雄激素结合蛋白的结构和功能

• 批准号: 5212527
• 负责人: GLEN L GUNSALUS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5212527
• 关键词: X ray crystallography; androgen binding protein; cell membrane; dimer; epididymis; genetic manipulation; glycoproteins; glycosylation; laboratory rabbit; laboratory rat; male reproductive system; mutant; protein structure function; site directed mutagenesis; transfection /expression vector

Rat androgen-binding protein (ABP), a secretory product of the Sertoli cell, is released primarily into seminiferous tubular lumen and transported to the epididymis where a portion of it is internalized. It is now known to belong to a family of extracellular androgen-binding proteins, which are synthesized in various organs under different regulation, and were originally thought to be distinct, but are, within a species, the product of a single gene. The high affinity and specificity of steroid binding lead to the view that it is central to the biologic function of these molecules. The recent finding that they can also bind to selected cells and, depending on the target tissue, stimulate cAMP accumulation or be internalized or both, suggests they may play a role in androgen delivery or subserve a signaling function. The observation that binding of steroid to these proteins can mediate their interaction with cellular receptors suggests a coupling of these two events, possibly through a conformational change in the protein upon steroid binding. Although these protein are known to be homodimers binding one mole of steroid per mole of native dimer, and the primary amino acid sequences for several of them have been determined, little is known about their physical structure or the nature of the steroid- or cell-recognition sites or how they may be coupled. Our goal is to relate these known actions to structure using site-directed or how they may be coupled. Our goal is to relate these known actions to structure using site-directed mutagenesis to probe the steroid binding site, the cell recognition domain, and the influence of glycosylation on biological activity. To accomplish this we will construct appropriate expression vectors for production of native and mutated ABPs in transient transfection studies and in stably transformed cells and assess their ability to bind steroid and to bind and stimulate target cells. While this approach will further delineate sequences involved in steroid and cellular binding, an understanding of how these regions interact and are related to each other spatially depends on elucidation of the tertiary and quaternary structure of ABP. To this end we will produce non-glycosylated ABP in quantities sufficient to do crystallization studies with the ultimate aim of determining its three-dimensional structure.

大鼠雄激素结合蛋白（ABP），支持细胞的分泌产物 细胞，主要释放到生精小管腔和运输 到附睾的一部分被内化。 现在已知 属于细胞外雄激素结合蛋白家族， 在不同的器官中以不同的调节方式合成， 最初被认为是不同的，但在一个物种中， 一个单一的基因。 类固醇结合的高亲和力和特异性 导致了这样一种观点，即它是这些生物功能的核心， 分子。 最近的发现是它们也可以与选定的细胞结合， 并且，根据靶组织，刺激cAMP积累或 内化或两者兼而有之，表明它们可能在雄激素递送中起作用， 有助于信号功能。 观察到类固醇与 这些蛋白质可以介导它们与细胞受体的相互作用 表明这两个事件的耦合，可能通过构象 类固醇结合后蛋白质的变化。 虽然这些蛋白质 已知是每摩尔天然类固醇结合一摩尔类固醇的同二聚体， 二聚体，其中几个的主要氨基酸序列已经被 确定，很少有人知道他们的物理结构或性质， 类固醇或细胞识别位点或它们如何偶联。 我们 我们的目标是将这些已知的动作与结构相关联， 它们是如何结合的。 我们的目标是将这些已知的行为与 结构使用定点诱变来探测类固醇结合 位点、细胞识别结构域以及糖基化对 生物活性 为了实现这一点，我们将建立适当的 用于瞬时表达天然和突变的ABP的表达载体 转染研究和稳定转化的细胞，并评估其 结合类固醇并结合和刺激靶细胞的能力。 虽然这 这种方法将进一步描述参与类固醇和细胞的序列， 结合，了解这些区域如何相互作用，并与 彼此在空间上取决于第三和第四的阐明 ABP的结构。 为此，我们将生产非糖基化ABP， 足以进行结晶研究的量，最终目的是 来确定它的三维结构。