Studies performed during the previous funding period of this Program Project have continued to provide significant new insights into cellular and molecular mechanisms that govern physiological processes in selected cell types in the kidney and urogenital tract. Based on these advances, and on the development of new tools and model systems during the previous funding period, Project 7 will define the complex intracellular recycling pathways of AQP2, identify protein interactions involved in vasopressin-induced trafficking, and explore novel cAMP-independent signaling pathways that can be used to bypass the vasopressin receptor in vivo in nephrogenic diabetes insipidus. Project 6 takes advantage of the development of cPLA2 knockout mice and cell lines to define the role of signaling via PLA2 and associated proteins in cellular proliferation, apoptosis and vesicle trafficking. The key role of an accessory protein, PLIP, in cytosol to nuclear signaling and import will be of considerable importance to these studies. Project 9 identified unsuspected functions for G-proteins and their regulating proteins, RGS proteins, in Golgi vesicle trafficking and in vesicle coatomer (COP) recruitment. The new studies will define in great detail the acidification-dependent and G-protein-dependent recruitment of vesicle coat proteins in the receptor-mediated endocytotic pathway of proximal tubules in vivo and in vitro. These studies will help determine the molecular mechanisms by which defective endosomal acidification results in proximal tubule reabsorptive dysfunction and Fanconi-syndrome. Project 12 provided the first definition of the cellular proteins and mechanisms of luminal acidification in the epididymis and vas deferens, a process that is critical for sperm maturation and storage. The new studies will examine cell and molecular interactions among selected membrane transport proteins and accessory proteins, and will define the short- and long-term hormonal regulation of acid base transport in this epithelium. The previous period of funding saw an exceptionally high level of interaction among the individual members of this Program Project, due to their complementary expertise in areas of cell biology, molecular biology, physiology and biochemistry, and their related interest in relating fundamental processes of cell signaling, protein trafficking and membrane function to whole organ physiology. The centralized Core - Microscopy facility has once again been a major factor in coordinating cell biological and morphological studies for all projects. We anticipate that the individual and collective efforts proposed in this renewal application will once again lead to a greater understanding of the relationship between basic cellular processes and normal function and disease states in urogenital cells and tissues.
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