Structure and characterization of the mouse UT-A gene (Slc14a2)

Abstract

The movement of urea across plasma membranes is modulated by facilitated urea transporter proteins. These proteins are the products of two closely related genes, termed UT-A ( Slc14a2) and UT-B ( Slc14a1). By genomic library screening and P1 artificial chromosome “shotgun” sequencing, we have determined the structure of the mouse UT-A gene. The gene is >300 kb in length, contains 24 exons, and has 2 distinct promoters. Flanking the 5′-region of the gene is the UT-Aα promoter that regulates transcription of UT-A1 and UT-A3. The second promoter, termed UT-Aβ, is present in intron 13 and regulates transcription of UT-A2. cAMP agonists (100 μM dibutryl cAMP, 25 μM forskolin, 0.5 mM IBMX) increased the activity of a 2.2-kb UT-Aα promoter construct 6.2-fold [from 0.026 ± 0.003 to 0.160 ± 0.004, relative light units (RLU)/μg protein] and a 2.4-kb UT-Aβ promoter construct 9.5-fold (from 0.020 ± 0.002 to 0.190 ± 0.043 RLU/μg protein) above that in untreated controls. Interestingly, only the UT-Aβ promoter contained consensus sequences for CREs and deletion of these elements abolished cAMP sensitivity. Increasing the tonicity of culture medium from 300 to 600 mosmol/kgH2O with NaCl caused a significant increase (from 0.060 ± 0.004 to 0.095 ± 0.010 RLU/μg protein) in UT-Aα promoter activity but had no effect on the UT-Aβ promoter. A tonicity-responsive enhancer was identified in UT-Aα and is suggested to be responsible for mediating this effect. Levels of UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with control animals, indicating that the activities of both promoters are likely to be elevated during prolonged antidiuresis.