J Biol Chem, Vol. 275, Issue 10, 7280-7288, March 10, 2000

Cloning and Characterization of the Promoter Region of the Rat Epidermal Growth Factor Receptor Gene and Its Transcriptional Regulation by Nerve Growth Factor in PC12 Cells^*

Xu-Wen Liu, Yasuhiro Katagiri, Hao Jiang, Li-Jie Gong^§, Li-Ying Guo, Makoto Shibutani^¶, Alfred C. Johnson^**, and Gordon Guroff

From the Section on Growth Factors and the ^§ Laboratory of Developmental and Molecular Immunity, NICHD, and the  Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892

Our previous studies have shown that treatment of PC12 cells with nerve growth factor (NGF) causes a profound down-regulation of the epidermal growth factor receptor (EGFR) mRNA and protein. Further, the NGF-induced down-regulation of the EGFR is under transcriptional control. To elucidate the molecular mechanism of this down-regulation we have cloned a 2.7-kilobase sequence from the promoter region of the rat EGFR from a rat P1 library. Six transcriptional 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 gene. This sequence exhibited functional promoter activity in transient transfection 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 for the down-regulation of the EGFR by NGF. Supportive evidence for the relevance of this sequence was obtained from gel mobility shift assays and by transfection of TCC mutation constructs. Our results demonstrate that TCC repeat sequences are required for the down-regulation of rat EGFR by NGF in PC12 cells and may lead to the identification of the NGF-responsive transcription factors.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF142153.

This article is dedicated to the memory of Dr. Gordon Guroff, Chief of Section on Growth Factors and Deputy Scientific Director of the NICHD. During his long career, he enriched the field of neuroscience and all of us who had the privilege to know and work with him.