Functional characterization of rat gp600/megalin promoter: combination of proximal Sp1 site and JCV repeat is important in rat gp600/megalin promoteractivation

Gp600/megalin is an endocytic receptor belonging to the low-density lipopro tein receptor family. Up or down regulation of this protein were observed i n certain disease states. To understand the mechanisms that control gp600/m egalin gene expression, we cloned and functionally characterized a 738-bp f ragment of the 5'-flanking region of rat gp600/megalin gene. A transcriptio n start site was mapped to 33 bp downstream of TAGAAA sequence (TATA-like b ox). Multiple transcription factor binding sites: were identified. Serial 5 ' deletions and transient transfection assays showed that the deletion frag ment containing the Spl site proximal to the TATA-like box and a JCV repeat retained 80% of the promoter activity. Individual mutations of the proxima l Sp1 site and JCV repeat reduced the promoter activity by 60 and 34% respe ctively. Double mutations of the proximal Sp1 site and JCV repeat produced a dramatic 80% reduction in the promoter activity. However, deletions and m utations or double mutations of other transcription factor binding sites in the promoter region had a minor effect on the promoter activity. These res ults indicate that the combination of proximal Spl site and the JCV repeat are necessary for activation of gp600/megalin expression. Moreover, Spl and Sp3 proteins interacted with the proximal and the distal Spl sites in the nuclear extracts of gp600/megalin expressing cell lines. TCF site seems to be involved in negative regulation of this promoter but no nuclear protein( s) were found to bind to this site. In addition, Ap2 site responsible for 2 8% promoter activity is able to bind two dominant unknown nuclear proteins. This functional characterization of the regulation of gp600/megalin gene i s likely to advance the knowledge of the regulation of this gene in health and disease. (C) 2001 Elsevier Science B.V. All rights reserved.