During the past decade, we and others have made tremendous progress in understanding the long-term regulation of urea transport proteins. The overall goals of the next funding period are to investigate whether vasopressin increases the apical plasma membrane accumulation of UT-A1, to determine the vasopressin- stimulated signaling pathway(s) that increase UT-A1 accumulation, and to study the retrieval, turnover, and degradation of UT-A1. Urea and water permeabilities often change together, but there are situations when they are regulated independently of one another in the IMCD. Our proposed studies will provide insights into an intriguing biological question: how does vasopressin, acting through a single receptor, the V2- receptor, regulate urea permeability independently of water permeability in a single nephron segment, the terminal IMCD and, indeed, probably independently within the same cells? HYPOTHESIS I. Vasopressin rapidly increases UT-A1 accumulation in the apical plasma membrane.
Specific Aim 1. Determine whether vasopressin increases UT-A1 accumulation in the apical plasma membrane through the V2-vasopressin receptor. Rationale: our preliminary data show that acute vasopressin administration increases the amount of UT-A1 in the plasma membrane.
Specific Aim 2. Determine which cyclic AMP pathway regulates UT-A1 accumulation in the plasma membrane. Rationale: our Preliminary Data show that activation of Epac, a novel cAMP-dependent but PKA-independent pathway, increases UT-A1 accumulation in the IMCD plasma membrane, but does not change AQP2. These data suggest that vasopressin's ability to independently regulate urea and water results, at least in part, from activation of different cAMP-dependent signaling pathways in IMCD cells. HYPOTHESIS II. Vasopressin regulates UT-A1 retrieval and/or degradation.
Specific Aim 3. Determine the functional half-life of UT-A1 in the apical plasma membrane and the cellular systems responsible for UT-A1 retrieval and/or degradation. Rationale: our Preliminary Data in UT-A1- MDCK cells show that: 1) UT-A1 is a ubiquitinated protein in rat inner medulla and in UT-A1-MDCK cells; and 2) pre-treatment with a proteasome inhibitor produces a marked increase in urea flux (function), UT-A1 protein abundance, and the sizes of biotinylated UT-A1. These data suggest that ubiquitination may regulate retrieval of UT-A1 from the plasma membrane and target it for degradation in the proteasome.
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