PROTEIN KINASE A ANCHORING IN SPERMATOZOAN FUNCTION

蛋白质激酶 A 锚定精子功能

• 批准号: 2889501
• 负责人: DANIEL W CARR
• 金额: $ 14.43万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2889501
• 关键词: binding proteins; biological signal transduction; calcium ion; cyclic AMP; electron microscopy; enzyme structure; immunocytochemistry; isozymes; laboratory rabbit; northern blottings; phosphorylation; protein kinase A; protein localization; protein sequence; protein structure function; sperm; sperm motility; western blottings; yeast two hybrid system

Efforts to develop safe and effective methods to regulate human reproduction, both infertility and contraception, would be greatly enhanced by a more complete understanding of the regulatory mechanisms which control sperm functions. The long term goal of this proposal is to develop a sperm targeted contraceptive based on the interruption of sperm specific signaling pathway. There is ample evidence that cAMP and the cAMP-dependent protein kinase (PKA) are involved in the regulation of sperm motility. PKA is a multi-functional enzyme with a broad substrate specificity. Recent studies show that subcellular targeting of PKA is an important, if not essential, requirement for cAMP action. This targeting of PKA is mediated by binding of the regulatory subunits (R) with A-kinase anchoring proteins (AKAPs). If localized PKA action is important in somatic cells, such a mechanism is likely to be even more critical in the highly compartmentalized sperm cell. We and others have shown that the type II alpha (RIIalpha) isoform is detected exclusively in the flagellum suggesting an involvement in sperm motility regulation. On the other hand, the type I isoforms are associated mostly with the apical and equatorial segments of the sperm head, suggesting their involvement in acrosome reaction and/or sperm-egg fusion. This specific PKA localization to distinct subcellular regions are likely to be mediated by AKAPs. Bovine, mouse, monkey and human sperm all contain one predominant RIIalpha binding AKAP with Mr of approximately 110,000. Purification and partial amino acid sequences show that sperm AKAP 110 is a novel protein. We have also shown that membrane permeable peptides, designed to disrupt the interaction of RIIalpha with sperm AKAPs, arrest sperm motility. We hypothesize that PKA anchoring is essential for sperm motility and fertilization. The overall goal of this proposal is to define the role of PKA anchoring in sperm function. Specifically, we will determine the structure and function of AKAP 110, document the subcellular location of PKA regulatory subunits and their AKAPs and define the key biochemical mechanisms regulated by PKA/AKAP110 interaction. Studies outlined in this proposal will facilitate the design new and highly specific pharmacological reagents for inhibiting sperm function in vivo. Knowledge gained from these studies may also be useful for the diagnosis and treatment of patients with defects in sperm movement and fertility.

努力开发安全有效的方法来调节人类 生殖，不孕不育和避孕，将大大 通过更全面地了解监管机制， 控制精子的功能本提案的长期目标是 开发一种基于精子阻断的精子靶向避孕药 特定的信号通路。有充分的证据表明，cAMP和 cAMP依赖性蛋白激酶（PKA）参与调节 精子活力PKA是一种底物广泛的多功能酶 的特异性最近的研究表明，PKA的亚细胞靶向是一种新的靶向治疗方法。 这是cAMP作用重要（如果不是必需）要求。该靶向 PKA的合成是通过调节亚基（R）与A激酶的结合来介导的 锚定蛋白（AKAP）。 如果局部PKA作用在体细胞中是重要的，那么这种机制是 在高度分隔的精子中可能更为关键 cell.我们和其他人已经表明，II型α（RIIalpha）亚型 仅在鞭毛中检测到， 精子运动调节另一方面，I型同种型是 主要与精子的顶端和赤道段有关 头部，表明它们参与顶体反应和/或精卵 核聚变这种特异性PKA定位于不同的亚细胞区域， 可能是由AKAP介导的。 牛、小鼠、猴和人的精子都含有一种主要的RII α 将AKAP与约110，000的Mr结合。纯化及部分 氨基酸序列表明精子AKAP 110是一种新蛋白。我们有 还表明，膜渗透肽，旨在破坏 RII α与精子AKAP相互作用，抑制精子运动。我们 假设PKA锚定对于精子运动是必需的， 受精本提案的总体目标是界定以下方面的作用： PKA锚定精子功能具体来说，我们将确定 AKAP 110的结构和功能，记录了AKAP 110的亚细胞位置。 PKA调节亚基和它们的AKAP，并定义了 PKA/AKAP 110相互作用的调节机制。 本提案中概述的研究将促进新的设计， 用于抑制精子功能的高度特异性药理学试剂， vivo.从这些研究中获得的知识也可能有助于 精子运动缺陷患者的诊断和治疗， 生育