EMORY UNIVERSITY
Grant: $100,000 - National Institutes of Health - Aug. 20, 2009
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Award Description: REGULATION OF RAT RENAL INNER MEDULLARY FUNCTION. The major goal of the parent grant, 5R01DK041707, is to test two hypotheses: 1?? vasopressin rapidly increases UT-A1 accumulation in the apical plasma membrane; and 2?? vasopressin regulates UT-A1 retrieval and/or degradation. We planned to test these hypothesis using native rat inner medullary collecting duct ??IMCD?? suspensions and MDCK cells that we stably transfected with UT-A1 ??UT-A1-MDCK cells??. Our studies to date have been very successful. We showed that vasopressin increases the plasma membrane accumulation of the UT-A1 urea transporter in rat IMCD suspensions, and in the apical plasma membrane of UT-A1-MDCK cells, which are dog in origin. We then showed that UT-A1 phosphorylation at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation. These findings raise important questions regarding the role of UT-A1 phosphorylation by vasopressin in regards to UT-A1 apical plasma membrane accumulation. There are many more molecular tools available for the mouse. Therefore, the major goal of this supplement is to stably transfect UT-A1 into mIMCD3 cells, which are mouse in origin. These cells should greatly accelerate the pace of discovery since we would be able to take advantage of the wide array of molecular reagents available in the mouse. We will also label UT-A1 with a fluorescent protein and stably transfect this into mIMCD3 cells which could be used to elucidate the intracellular trafficking of UT-A1. The parent grant proposed studies of UT-A1 phosphorylation. However, it was not known that there were two serines in UT-A1 that are phosphorylated by vasopressin. To determine the role of each phosphorylation, we propose to make stably transfected mIMCD3 cell lines with S486 or S499 phospho-mutant or phospho-mimetic forms of UT-A1. To further advance our work and allow us to examine the individual phosphorylations in rat IMCD, we need S486 and S499 phospho-specific antibodies. We have preliminary data showing that we have made a UT-A1-S486-specific phospho-antibody, and we will use a similar approach to make a UT-A1-S499 phospho-specific antibody. Specific Aim 1. To generate stably transfected mIMCD3 cell lines expressing S486 or S499 phospho-mutant or phospho-mimetic forms of UTA1. Specific Aim 2. To generate stably transfected mIMCD3 cell lines expressing fluorescently-labeled UT-A1.
Project Description: Specific Aim 1. To generate stably transfected mIMCD3 cell lines expressing S486 or S499 phospho-mutant or phospho-mimetic forms of UT-A1. Specific Aim 2. To generate stably transfected mIMCD3 cell lines expressing fluorescently-labeled UT-A1. To further advance our work and allow us to examine the individual phosphorylations in rat IMCD suspensions, we need S486 and S499 phospho-specific antibodies. We have begun charadcterizing a UT-A1-S486-specific phospho-antibody. This antibody seems to preferentially recognize the 117 kDa form of UT-A1. As a control, we have compared the phospho antibody to P32 labelled immunoprecipitations and the correlation is excellent. The antibody works for westerns and immunohistochemistry in rat and mouse inner medulla. We have begun work on a UT-A1-S499 phospho-specific antibody, but no test bleeds are yet available. We have also prepared a stably transfected S486A/S499A UT-A1 mIMCD3 cell line and have begun characterizing this new line.
Infrastructure Description: N/A
Jobs Summary: Retention of one full-time research specialist, Mr. Chad Denson. Mr. Denson was hired in December 2008 to work on another grant that is now in a no-cost extension. He has gained experience with the techniques proposed in this supplement. Mr. Denson's current position was to have terminated May 31, 2009. This supplement enabled Mr. Denson's position to continue. Mr. Denson is responsible for transfecting the cells, growing the cells in culture, maintaining the cell lines, and performing the western blots, all under the direction of Dr. Sands (PI) and Dr. Klein (co-PI of the parent grant). Mr. Denson will also be responsible for providing these cells and our antibodies to other investigators who request them. (Total jobs reported: 0)
Project Status: Less Than 50% Completed
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