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

描述（由申请人提供）：所有细胞的鞭毛和纤毛均受环腺苷酸（cAMP）依赖性蛋白激酶（PKA）调节，但该调节机制尚不清楚。PKA通过PKA的调节（R）亚基上的二聚化/对接结构域与A-激酶锚定蛋白（AKAP）相互作用而靶向。我们已经确定了四种新的哺乳动物蛋白，共享PKA的RII二聚化/对接（R2D2）结构域，因此也结合AKAP。其中两种蛋白（ASP和ROPN1）与AKAP以类似于PKA的方式相互作用;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。AKAP 3和4、PKA和ROPN 1都位于精子的纤维鞘（FS）中。FS是鞭毛细胞骨架结构 是精子特有的，在鞭毛的主要部分包围着外部致密的纤维和轴丝。最初被认为主要作为鞭毛的支持结构，我们现在知道FS通过作为糖酵解和信号酶如PKA的支架在运动调节中起着关键作用。在实验室和临床上，FS相关蛋白的破坏与弱精子症和生育能力受损可靠相关。虽然很少有遗传因素与人类男性不育直接相关，但对于这项工作来说重要的是，PKA，AKAP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ASP和/或ROPN 1的转基因小鼠品系的研究。使用这些模型，我们已经证明，ROPN 1的缺乏导致雄性小鼠中进行性运动精子的百分比显著降低和生育能力受损。在ASP和ROPN 1都不存在的情况下，雄性由于鞭毛的主要部分的结构缺陷而不育，该结构缺陷似乎包含在纤维鞘（FS）中，导致完全不运动。我们推测ROPN1通过锚定AKAP参与精子发生过程中纤维鞘的形成。为了验证这一假设，我们将描述这些蛋白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的结合是如何调节的。该提案的目标是确定ROPN1/ASP在纤维鞘的发育和功能中的作用。增加对FS中必需蛋白质相互作用的了解将为男性弱精子症和不育症提供深入了解。我们最近证明，缺乏ASP的小鼠表现出降低的基础纤毛搏动频率在支气管，因此，我们的研究结果也可能在纤毛功能和疾病的意义。我们实验室的长期目标是阐明ASP和ROPN 1在纤毛和鞭毛运动调节中的功能，最终目标是开发男性避孕药和治疗涉及粘膜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.