Identification of a novel glucocorticoid receptor mutation in budesonide-resistant human bronchial epithelial cells.
We developed a molecular genetic model to investigate glucocorticoid receptor (GR) signaling in human bronchial epithelial cells in response to the therapeutic steroid budesonide. Based on a genetic selection scheme using the human Chago K1 cell line and integrated copies of a glucocorticoid-responsive herpes simplex virus thymidine kinase gene and a green fluorescent protein gene, we isolated five Chago K1 variants that grew in media containing budesonide and ganciclovir. Three spontaneous budesonide-resistant subclones were found to express low levels of GR, whereas two mutants isolated from ethylmethane sulfonate-treated cultures contained normal levels of GR protein. Analysis of the GR coding sequence in the budesonide-resistant subclone Ch-BdE5 identified a novel Val to Met mutation at amino acid position 575 (GRV575M) which caused an 80% decrease in transcriptional regulatory functions with only a minimal effect on ligand binding activity. Homology modeling of the GR structure in this region of the hormone binding domain and molecular dynamic simulations suggested that the GRV575M mutation would have a decreased affinity for the LXXLL motif of p160 coactivators. To test this prediction, we performed transactivation and glutathione-S-transferase pull-down assays using the p160 coactivator glucocorticoid interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 and found that GRV575M transcriptional activity was not enhanced by GRIP1 in transfected cells nor was it able to bind GRIP1 in vitro. Identification of the novel GRV575M variant in human bronchial epithelial cells using a molecular genetic selection scheme suggests that functional assays performed in relevant cell types could identify subtle defects in GR signaling that contribute to reduced steroid sensitivities in vivo.
Kunz
Susan
S
Department of Biochemistry and Molecular Biophysics, 1041 East Lowell Street, University of Arizona, Tucson, Arizona 85721, USA.
Sandoval
Robert
R
Carlsson
Peter
P
Carlstedt-Duke
Jan
J
Bloom
John W
JW
Miesfeld
Roger L
RL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D001980:Bronchi / Q000502:physiology*; D019819:Budesonide / Q000494:pharmacology*; D002283:Carcinoma, Bronchogenic; D002460:Cell Line; D045744:Cell Line, Tumor; D004351:Drug Resistance; D015774:Ganciclovir / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms; D008715:Methionine; D008969:Molecular Sequence Data; D009092:Mucous Membrane; D020125:Mutation, Missense; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014633:Valine
D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D019819:Budesonide; D008715:Methionine; D005982:Glutathione Transferase; D014633:Valine; D015774:Ganciclovir