Cloning and characterization of the promoter region of the rat epidermal growth factor receptor gene and its transcriptional regulation by nerve growth factor in BC12 cells

Our previous studies have shown that treatment of PC12 cells with nerve gro wth factor (NGF) causes a profound down-regulation of the epidermal growth factor receptor (EGFR) mRNA and protein. Further, the NGF-induced down-regu lation of the EGFR is under transcriptional control. To elucidate the molec ular mechanism of this down-regulation we have cloned a 2.7-kilobase sequen ce from the promoter region of the rat EG;FR from a rat P1 library. Six tra nscriptional start sites were identified by 5'-rapid amplification of cDNA ends and primer extension. Sequence analysis showed a 62% overall homology with the human EGFR promoter region. To investigate its transcription, 1.1 kilobases of the 5'-flanking sequence were fused to a luciferase reporter g ene. This sequence exhibited functional promoter activity in transient tran sfection experiments with PC12, C6, and CV-1 cells. Treatment of PC12 cells with NGF inhibited promoter activity. By transfection of promoter deletion constructs, a silencer element was found between nucleotides -260 and -181 , and TCC repeat sequences appeared to be at least partially responsible fo r the down-regulation of the EGFR by NGF. Supportive evidence for the relev ance of this sequence was obtained from gel mobility shift assays and by tr ansfection of TCC mutation constructs. Our results demonstrate that TCC rep eat sequences are required for the down-regulation of rat EGFR by NGF in PC 12 cells and may lead to the identification of the NGF-responsive transcrip tion factors.