Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells.
The molecular basis for the receptorless (r-) and activation-labile (act1) phenotypes of glucocorticoid-resistant mutants isolated from glucocorticoid-sensitive human leukemic CEM-C7 cells was determined. Clones isolated from a complementary DNA library prepared from r- ICR27TK.3 cells, in which one glucocorticoid receptor (GR) gene has been deleted, contained a single adenosine to thymidine transversion in the third position of codon 753, resulting in the substitution of phenylalanine for leucine. This mutant gene (GR753F) had only 13% of the trans-activating activity of the normal gene and produced a M(r) 92,000 receptor protein with the same r- phenotype seen in ICR27TK.3 cells. Analysis of complementary DNA clones isolated from a library prepared from parental glucocorticoid-sensitive 6TG1.1 cells showed that these cells express both a normal GR gene (GR+) and the GR753F gene. Thus, their genotype is GR+/GR753F. Analysis of clones isolated from a complementary DNA library prepared from glucocorticoid-resistant activation-labile 3R7. 6TG.4 cells revealed the presence of the GR753F gene and a second mutant gene (GR421Y) containing a guanosine to adenosine transition in the second position of codon 421, resulting in the replacement of the first cysteine of the proximal zinc finger of the DNA-binding domain by tyrosine. This mutant had no trans-activating activity but normal ligand-binding characteristics. Thus, the genotype of act1 3R7.6TG.4 cells is GR421Y/GR753F. Consequently, the sequence-specific DNA-binding activity of receptors in act1 cells is attributable to the GR753F gene, while the ligand-binding activity seen in intact cells is attributable to the GR421Y gene. These results provide a direct explanation for the r- and act1 phenotypes of glucocorticoid-resistant cells and demonstrate that glucocorticoid-sensitive cells derived from CEM-C7 cells contain a heterogeneous population of normal and mutant receptors.
Powers
J H
JH
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Hillmann
A G
AG
Tang
D C
DC
Harmon
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003062:Codon / Q000737:chemistry / Q000235:genetics; D003907:Dexamethasone / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D017353:Gene Deletion; D005838:Genotype; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003062:Codon; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase