The role of R2D2/AKAP interactions in fibrous sheath function.

描述(申请人提供)：所有细胞的鞭毛和纤毛都受环磷酸腺苷(CAMP)依赖的蛋白激酶(PKA)的调节，尽管这种调节的机制尚不清楚。PKA通过其调节亚基(R)上的二聚化/对接结构域与A-激酶锚定蛋白(AKAP)相互作用而成为靶点。我们已经鉴定出四种新的哺乳动物蛋白，它们共享PKA的RII二聚化/对接(R2D2)结构域，因此也与AKAP结合。其中两个蛋白(ASP和ROPN1)以类似于PKA的方式与AKAP相互作用；然而，在对接结构域之外，R2D2蛋白与PKA几乎没有相似性，这表明它们具有不同的功能。AKAPs 3和4、PKA和ROPN1均位于精子的纤维鞘中。FS是一种鞭毛状细胞骨架结构 是围绕鞭毛主要部分的外部致密纤维和轴丝的精子所特有的。最初认为FS主要作为鞭毛的支撑结构，现在我们知道FS通过作为糖细胞和信号转导酶(如PKA)的支架在运动调节中发挥关键作用。在实验室和临床上，FS相关蛋白的中断可靠地与弱精子症和生育能力受损有关。虽然很少有遗传因素与人类男性不育直接相关，但对这项工作来说很重要的是，PKA、AKAPs和ROPN1表达的缺陷都与人类男性因素不育直接相关。我们最近发表了两项使用缺乏天冬氨酸转氨酶和/或ROPN1的转基因小鼠的研究。使用这些模型，我们已经证明，缺乏ROPN1会导致雄性小鼠逐渐运动的精子百分比显著降低，并导致生育能力下降。在没有ASP和ROPN1的情况下，雄性不育是由于鞭毛主段的结构缺陷，似乎包含在纤维鞘(FS)，导致完全静止。我们假设ROPN1通过在精子发生过程中锚定AKAP参与纤维鞘的形成。为了验证这一假设，我们将表征这些蛋白质在精子发生过程中的表达和定位，确定缺乏ASP/ROPN1的精子中存在的特定结构缺陷，并确定AKAP与PKA、ASP和ROPN1结合的调节方式。这项建议的目的是确定ROPN1/ASP在纤维鞘的发育和功能中的作用。增加对FS中基本蛋白质相互作用的了解将有助于深入了解男性的弱精子症和不育。我们最近证明，缺乏天冬氨酸的小鼠表现出支气管内基础纤毛搏动频率降低；因此，我们的结果可能对纤毛功能和疾病也有意义。本实验室的长期目标是阐明ASP和ROPN1在纤毛和鞭毛运动调节中的作用，最终目的是开发男性避孕药和治疗涉及粘液纤毛清除受损和男性因素不育的疾病。

DESCRIPTION (provided by applicant): Flagella and cilia of all cells are regulated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) though the mechanisms of this regulation are unclear. PKA is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. We have identified four novel mammalian proteins that share the RII dimerization/docking (R2D2) domain of PKA and therefore also bind to AKAPs. Two of these proteins (ASP and ROPN1) interact with AKAPs in a manner similar to PKA; however, outside the docking domain, R2D2 proteins bear little or no similarity to PKA suggesting they have distinct functions. AKAPs 3 and 4, PKA and ROPN1 are all located in the fibrous sheath (FS) of spermatozoa. The FS is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme in the principal piece of the flagella. Originally thought to function primarily as a support structure for the flagella, we now know that the FS plays a critical role in motility regulation by acting as a scaffold for glycolyti and signaling enzymes such as PKA. Disruptions in FS-associated proteins are reliably associated with asthenozoospermia and impaired fertility both in the laboratory and in the clinic. Though few genetic factors have been directly linked to human male infertility, importantly for this work, defects in PKA, AKAPs and ROPN1 expression have all been directly linked to male-factor infertility in humans. We have recently published two studies using genetically modified mouse lines that lack ASP and/or ROPN1. Using these models, we have demonstrated that lack of ROPN1 results in a significant reduction in the percentage of progressively motile sperm and impaired fertility in male mice. In the absence of both ASP and ROPN1, males are infertile due to structural defects in the principal piece of the flagellum that appear to be contained to the fibrous sheath (FS), resulting in complete immotility. We hypothesize that ROPN1 participates in fibrous sheath formation by anchoring AKAPs during spermatogenesis. To test this hypothesis, we will characterize the expression and localization of these proteins during spermatogenesis, identify specific structural defects present in sperm lacking ASP/ROPN1, and determine how AKAP binding to PKA, ASP and ROPN1 is regulated. The goal of this proposal is to determine the role of ROPN1/ASP in the development and function of the fibrous sheath. Increased knowledge of essential protein interactions in FS will provide insight into asthenozoospermia and infertility in men. We have recently demonstrated that mice lacking ASP exhibit reduced basal ciliary beat frequency in the bronchus; thus our results may also have significance in ciliary function and disease. The long-term goal of our laboratory is to elucidate the functions and ASP and ROPN1 in the regulation of ciliary and flagellar motility with the ultimate objective of developing male contraceptives and treatments for diseases involving impaired mucociliary clearance and male factor infertility.