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

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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.