Cyclic nucleotide regulation of Na+/glucose cotransporter (SGLT1) mRNA stability - Interaction of a nucleocytoplasmic protein with a regulatory domain in the 3 '-untranslated region critical for stabilization

Expression of the Nac-coupled glucose cotransporter SGLT1 is regulated post -transcriptionally at the level of mRNA stability. We have previously demon strated that cAMP-dependent stabilization of the SGLT1 message was correlat ed with the protein phosphorylation-dependent binding of cytoplasmic protei ns to a uridine-rich sequence (URE) in the 3'-untranslated region (UTR). In the present study, the regulatory role of the URE was demonstrated by inse rting it into the 3'-UTR of a beta -globin reporter minigene under the cont rol of the tetracycline-regulated promoter. The resultant chimeric globin/S GLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decre ased half-life compared with the beta -globin control, indicating that the URE serves a destabilizing function, Activation of protein kinase A stabili zed the chimeric message but not the beta -globin control, indicating the p resence of a regulatory stabilizing sequence within the URE. A 38-kDa nucle ocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE, A mutation in this binding site that prevented protei n binding assayed in vitro by UV cross-linking also prevented protein kinas e A-dependent stabilization of the chimeric message assayed in vivo, These findings identify the interaction between a 38-kDa nucleocytoplasmic protei n and a regulatory uridine-rich sequence in the 3'-UTR as critical for cAMP -mediated SGLT1 message stabilization.