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Updated: Aug 4, 2026
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic
J H Powers1, A G Hillmann, D C Tang
1Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Abstract:
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.
Insights
Glucocorticoid resistance in leukemia cells stems from specific gene mutations affecting the glucocorticoid receptor (GR). These mutations, GR753F and GR421Y, explain receptorless and activation-labile phenotypes, revealing a mix of normal and mutant receptors in sensitive cells.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Glucocorticoid resistance in human leukemic cells is a significant clinical challenge.
- Understanding the molecular basis of glucocorticoid receptor (GR) mutations is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying the receptorless (r-) and activation-labile (act1) phenotypes in glucocorticoid-resistant CEM-C7 cell mutants.
- To identify specific mutations in the glucocorticoid receptor gene responsible for these resistance phenotypes.
Main Methods:
- Isolation and analysis of complementary DNA (cDNA) libraries from glucocorticoid-resistant and sensitive cell lines.
- DNA sequencing to identify point mutations in the glucocorticoid receptor (GR) gene.
- Assessment of trans-activating and ligand-binding activities of mutant GR proteins.
Main Results:
- A single adenosine to thymidine transversion in codon 753 (GR753F) resulted in a receptorless (r-) phenotype with reduced trans-activating activity.
- A guanosine to adenosine transition in codon 421 (GR421Y) led to an activation-labile (act1) phenotype with no trans-activating activity but normal ligand binding.
- Glucocorticoid-sensitive cells expressed both normal GR and GR753F, indicating a heterogeneous receptor population.
Conclusions:
- The GR753F mutation explains the receptorless phenotype, while the GR421Y mutation explains the activation-labile phenotype in resistant cells.
- Sequence-specific DNA binding in act1 cells is mediated by GR753F, and ligand binding by GR421Y.
- CEM-C7 derived cells exhibit a mixed population of normal and mutant glucocorticoid receptors, contributing to varied resistance profiles.
