PROTEIN KINASE A ANCHORING IN SPERMATOZOAN FUNCTION

努力制定安全有效的方法来规范人类 生殖，无论是不孕不育还是避孕，都将大大 通过更全面地了解监管机制而得到加强 它控制着精子的功能。这项提议的长期目标是 基于精子阻断的精子靶向避孕药的研制 特定的信号通路。有充分的证据表明，坎普和 CAMP依赖的蛋白激酶(PKA)参与调节 精子活力。PKA是一种底物广泛的多功能酶。 专一性。最近的研究表明，亚细胞靶向PKA是一种 营地行动的重要要求，如果不是必要的话。这一目标 通过调节亚基(R)与A-激酶结合来调节PKA的活性 锚定蛋白(AKAP)。 如果局部的PKA作用在体细胞中是重要的，这样的机制是 在高度分隔的精子中可能更加关键 手机。我们和其他人已经证明了II型阿尔法(RIIpha)亚型 仅在鞭毛中检测到，表明参与了 精子运动调节。另一方面，I型异构体是 主要与精子顶端和赤道部分有关 头部，表明它们参与了顶体反应和/或精子-卵子 核聚变。这种特定的PKA定位于不同的亚细胞区域是 很可能是由AKAP调解的。 牛、小鼠、猴子和人类精子都含有一种主要的RIIpha 结合AKAP的MR约为110,000。提纯和部分 氨基酸序列分析表明，精子AKAP 110是一种新的蛋白质。我们有 还表明，膜通透性多肽，旨在破坏 RIIpha与精子AKAPs相互作用，抑制精子活力。我们 假设PKA锚定对精子活力和 受精。这项提案的总体目标是定义 PKA锚定在精子功能中。具体来说，我们将确定 AKAP 110的结构和功能，记录了 PKA调节亚基及其AKAPs及其关键生化的定义 PKA/AKAP110相互作用的调控机制。 这项建议中概述的研究将有助于设计新的和 抑制小鼠精子功能的高特异性药物 活着。从这些研究中获得的知识也可能对 精子运动和运动障碍患者的诊断和治疗 生育能力。

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.