Purification of a human glucocorticoid receptor gene promoter-binding protein. Production of polyclonal antibodies against the purified factor.
The glucocorticoid receptor (GR) is an essential protein involved in mediating glucocorticoid-regulated gene transcription. The cellular GR concentration is modulated by a number of factors including glucocorticoids, which are capable of down-regulating their own receptor concentration. To further study this phenomenon, the human GR (hGR) gene promoter was isolated and was shown to contain the sequences essential for glucocorticoid-dependent down-regulation in CV-1 cells by gene transfer. Further transfections performed with the hGR gene demonstrated that the nucleotide sequence between -250 and -750 is implicated in the down-regulation of the hGR by hormone. The promoter region of human GR is extremely rich in G+C sequences which are known to be involved in the regulation of many housekeeping genes such as GR, as well as a number of cellular oncogenes. Using a combination of partial purification of DNA-binding proteins, DNA-protein interaction by gel shift analysis and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have identified a protein factor (GRF-1) of 95 kDa which interacts with the human GR gene fragment implicated in homologous down-regulation. Polyclonal antibodies were then raised against the protein following purification. This method of purification and identification may be universally applied to purify and characterize unknown factors which may be involved in the regulation of genes such as hGR gene.
Leclerc
S
S
Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, Quebec, Canada.
Xie
B X
BX
Roy
R
R
Govindan
M V
MV
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D015536:Down-Regulation; D019204:GTP-Binding Proteins; D005786:Gene Expression Regulation; D005796:Genes; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D012097:Repressor Proteins; D015183:Restriction Mapping; D014162:Transfection; D025521:Tumor Suppressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1
C074560:ARHGAP35 protein, human; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009841:Oligonucleotides; C513039:RASGRF1 protein, human; D011965:Receptors, Glucocorticoid; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D019204:GTP-Binding Proteins