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

Gp600/megalin is an endocytic receptor belonging to the low-density lipoprotein receptor family. Up or down regulation of this protein were observed in certain disease states. To understand the mechanisms that control gp600/megalin gene expression, we cloned and functionally characterized a 738-bp fragment of the 5'-flanking region of rat gp600/megalin gene. A transcription start site was mapped to 33 bp downstream of TAGAAA sequence (TATA-like box). Multiple transcription factor binding sites were identified. Serial 5' deletions and transient transfection assays showed that the deletion fragment containing the Sp1 site proximal to the TATA-like box and a JCV repeat retained 80% of the promoter activity. Individual mutations of the proximal Sp1 site and JCV repeat reduced the promoter activity by 60 and 34% respectively. Double mutations of the proximal Sp1 site and JCV repeat produced a dramatic 80% reduction in the promoter activity. However, deletions and mutations or double mutations of other transcription factor binding sites in the promoter region had a minor effect on the promoter activity. These results indicate that the combination of proximal Sp1 site and the JCV repeat are necessary for activation of gp600/megalin expression. Moreover, Sp1 and Sp3 proteins interacted with the proximal and the distal Sp1 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 28% promoter activity is able to bind two dominant unknown nuclear proteins. This functional characterization of the regulation of gp600/megalin gene is likely to advance the knowledge of the regulation of this gene in health and disease.