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 Na(+)-coupled glucose cotransporter SGLT1 is regulated post-transcriptionally at the level of mRNA stability. We have previously demonstrated that cAMP-dependent stabilization of the SGLT1 message was correlated with the protein phosphorylation-dependent binding of cytoplasmic proteins 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 inserting it into the 3'-UTR of a beta-globin reporter minigene under the control of the tetracycline-regulated promoter. The resultant chimeric globin/SGLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decreased half-life compared with the beta-globin control, indicating that the URE serves a destabilizing function. Activation of protein kinase A stabilized the chimeric message but not the beta-globin control, indicating the presence of a regulatory stabilizing sequence within the URE. A 38-kDa nucleocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE. A mutation in this binding site that prevented protein binding assayed in vitro by UV cross-linking also prevented protein kinase A-dependent stabilization of the chimeric message assayed in vivo. These findings identify the interaction between a 38-kDa nucleocytoplasmic protein and a regulatory uridine-rich sequence in the 3'-UTR as critical for cAMP-mediated SGLT1 message stabilization.