Renal proximal tubule and intestinal brush border membranes contain SGLT family transporters that catalyze the Na+ coupled secondary active transport of glucose. Two distinct Na+/glucose cotransporter isoforms, SGLT1 and SGLT2, are expressed in the LLC-PK1 renal epithelial cell line, and SGLT1 expression is regulated by cell density and by the action of protein kinases A and C. The applicant has identified a post-transcriptional mechanism for the regulation of SGLT1 mRNA stability that may play a key role in modulating the levels of this transporter in response to cell signalling mechanisms. Protein kinase A activation promotes SGLT1 message stabilization, which is an effect that requires the 3' untranslated region (3'-UTR) and is associated with protein phosphorylation as well as the increased binding of a 48 kDa protein (termed SG-URBP) to a uridine-rich element (URE) in the SGLT1 3' UTR. The present application will further characterize possible cis- and trans-acting factors that mediate post-transcriptional regulation of cotransporter expression. Using chimeric mRNA constructs, Specific Aim 1 will test the hypothesis that the SGLT1 URE sequence located in the 3' UTR regulates SGLT1 message stability and efforts will be made to identify sequence elements involved in message stabilization by protein kinase A activation and SG-URBP binding.
In Specific Aim 2, the molecular mechanism of protein kinase C-mediated destabilization of the SGLT1 message will be explored by identifying cis-acting sequences and their cognate mRNA binding proteins.
In Specific Aim 3, SG-URBP will be purified and antibodies generated against it. These reagents should facilitate experiments focused on examining the influence of protein kinase A-mediated SG-URBP phosphorylation on its mRNA binding activity, and may aid in SG-URBP cloning and localization studies. Finally, in Specific Aim 4, the mechanisms that regulate SGLT2 expression will also be characterized.
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