Recombination of endogenous D2 dopamine receptor gene with a metallothionein promoter in GH4C1 cells confers functional and inducible D2 response.
We have previously shown that expression of a functional endogenous D2 short dopamine receptor is obtained in GH4C1 cells following transfection with a plasmid that confers resistance to neomycin (pRSVNeo) (Allard et al. (1993) Biochem. Biophys. Res. Commun. 193, 801-807). In order to better understand the mechanisms responsible for such a phenomenon, we cloned and sequenced the 5' region of the D2 gene present in native GH4C1 cells as well as the cDNA of transfected cells. No homology with the published sequence of the rat D2 dopamine receptor promoter was found; however, this region has perfect homology with the mouse metallothionein promoter. In cells expressing D2 receptor, the promoter is fully functional and can regulate dopaminergic D2 receptor mRNA levels and receptor expression in a dose-dependent manner in the presence of Zn2+ or Cd2+. The receptor level is raised from 500 to 3000 fmol/mg of protein in the presence of 100 microM of Zn2+. These results suggest that in GH4C1 cells, a recombination between the mouse metallothionein promoter and the D2 dopamine receptor took place. This system provides us with a cell line expressing an endogenous dopamine D2 receptor in which the level of expression can be easily modulated.
Allard
S
S
Unité de Médecine Génétique et Moléculaire, CHUL, Ste.-Foy, Quebec, Canada.
Labbé
M
M
Falardeau
P
P
D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000187:drug effects; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000235:genetics*; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology
D017931:DNA Primers; D012333:RNA, Messenger; D017448:Receptors, Dopamine D2; D002104:Cadmium; D008668:Metallothionein; D015032:Zinc