Abstract

The low-affinity nerve growth factor receptor (LNGFR) is a membrane-associated glycoprotein which is thought to participate in some of the biological activities of nerve growth factor (NGF). Expression of the LNGFR gene is known to be regulated both during development and in response to various agents in cell culture. However, molecular mechanisms responsible for the regulation have not been described. We report here an analysis of a 4.8-kb sequence from the 5′-flanking region of the rat LNGFR gene. Several regulatory elements were identified m this region by transfection of plasmid constructs containing sequences from LNGFR fused to a bacterial cat reporter gene. The proximal part of the promoter region (0.4-kb) was shown to be sufficient to support cat expression m all cell types used. A silencer element located between −1.5 kb and −1.8 kb from the start of translation, as well as an enhancer element in more upstream regions of the promoter, were identified in the phaeochromocytoma cell line, PC 12, and in the Sertoli cell line, TM4, that express the LNGFR gene. Treatment of TM4 cells with retinoic acid (RA) increases the level of LNGFR mRNA twofold, while testosterone treatment results in a tenfold decrease. Regions of the promoter responsive to testosterone and RA in TM4 cells were found at −610 to −860 bp and −1840 to −4800 bp upstream from the translation start codon, respectively. A RA-responsive element active in PC12 cells is located between bp −610 to −860 from the start codon.

Keywords: Gene cloning; Sertoli cells; PC12 cells; enhancer; silencer; CAT assay

Abbreviations: BDNF, brain-derived neurotrophic factor; bp, base pair(s); CAT, chloramphenicol acetyl transferase; cat, CAT-encoding gene; cDNA, DNA complementary to RNA; cpm, counts per minute; DMEM, Dulbecco's modified Eagle's medium; HNGFR, high affinity NGFR; kb, kilobase(s) or 1000 bp; LNGFR, low affinity NGFR; LNGFR, gene encoding LNGFR; NGF, nerve growth factor; NGF, gene encoding NGF; NGFR, NGF receptor; nt, nucleotide(s); NT-3, neurotrophin-3; NT-4, neurotrophin-4; RA, retinoic acid; TK, thymidine kinase; TK, TK-encoding gene