System B⁰ is a sodium-dependent, neutral amino acid transporter with broad specificity. The cloned transporter hATB⁰ generates system B⁰-like activity (Kekuda et al. 1996) and is regulated by epidermal growth factor (EGF) (Torres-Zamorano et al. 1997). We describe here the regulation of hATB⁰ by EGF in COS-7 cells.

Cells were grown for 4 days in control medium, with or without 100 ng ml^-1 EGF for the last 7 h of growth. Total RNA was extracted and hATB⁰ mRNA levels were measured by semi-quantitative RT-PCR, normalised against 18S rRNA levels. Compared with control, hATB⁰ mRNA was increased upon EGF supplementation by 1.63 ± 0.23f-fold (mean ± S.E.M., n = 8, P < 0.05, Student’s paired t test).

The promoter region of hATB⁰ was sequenced. Use of the Basic Local Alignment Search Tool (BLAST) available at the National Center for Biotechnology Information, revealed a 100 % match at the 5â end of the hATB⁰ cDNA (accession number U53347) with a CpG island clone (Cross et al. 1994). The clone was sequenced, which identified 170 bp of the promoter region of the hATB⁰ gene. BLAST searches using this promoter sequence identified a further 5 kb of the 5â-flanking region. Putative cis-acting binding sites were identified by use of the transcription factor matrix search programme MatInspector (Quandt et al. 1995). The promoter contains several GC-rich regions that bind the ubiquitous transcription factor Sp1, which has been shown to be involved in EGF-induced promoter activity.

A DNA fragment containing the region from -932 to +42 of the hATB⁰ gene, which includes six putative cis-acting Sp1 sites, was generated by PCR and inserted 5â to the coding region of the luciferase gene of the vector pGL3-basic (Promega), to create a reporter construct. Cells were transfected with reporter construct DNA by calcium phosphate co-precipitation and grown for 48 h in control medium, with or without 100 ng ml^-1 EGF for the last 7 h of growth. Cells were then harvested and assayed for luciferase activity. Compared with control, hATB⁰ promoter activity was increased by 1.37 ± 0.05-fold (n = 5, P<0.05) in the presence of EGF.

We conclude that the EGF-induced increase in hATB⁰ mRNA in COS 7 cells results from increased transcription and suggest that this effect is mediated through cis-acting Sp1 sites.

This work was supported by BBSRC.