CHARACTERIZATION OF THE HUMAN MEGALIN LRP-2 PROMOTER IN-VITRO AND IN PRIMARY PARATHYROID CELLS/

The gp330/Megalin/LRP-2 protein belongs to the low-density lipoprotein receptor gene family and is believed to function as an endocytic rece ptor for the uptake of lipoproteins and many other ligands, Other func tions of this protein may include a role in calcium sensing in the par athyroid glands and other tissues. In order to study the transcription al regulation of the human LRP-2 gene, a clone containing the 5'-flank ing region was isolated from a genomic DNA Library, and a transient tr ansfection protocol for primary bovine parathyroid cells was establish ed. RNA mapping techniques located the transcriptional start site 136 bp upstream of the initiation codon, Transient expression in several c ell types, including primary parathyroid cells, and in vitro transcrip tion in HeLa cell nuclear extracts showed that sequences between -120 and -35 were important for activated transcription. This region contai ns consensus binding sites (GC boxes) for transcription factor Sp1. Mu tation of the CC boxes abolished binding of Sp1 in vitro and resulted in reduced transcription in vitro and in transfected cells, Furthermor e, Sp1 stimulated transcription when tethered to the LRP-2 core promot er through a heterologous DNA-binding domain. Through site-directed mu tagenesis, we identified a novel atypical TATA element with the sequen ce TAGAAAA, Intriguingly, this sequence motif was shown previously not to mediate transcription in a systematic mutational analysis of the T ATA motif, Possible roles of this novel TATA element in the regulation of transcription initiation are discussed. The isolation and characte rization of the LRP-2 promoter and the 5'-flanking region and the esta blishment of a transient expression assay in primary parathyroid cells will facilitate studies on the regulatory mechanisms of the LRP-2 gen e and of other genes expressed in the parathyroid glands.