Identification of a Novel Glucocorticoid Receptor Mutation in Budesonide-Resistant Human Bronchial Epithelial Cells

Abstract

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.

Crosstalk between the glucocorticoid receptor and other transcription factors: Molecular aspects

“…Similarly, the Val residue 575 was predicted by molecular modeling to make contacts with the LxxLL motifs of co-activators. Accordingly, GR V575A lost its capacity to interact with GRIP1 in GST pull-down assays, but was still able to repress NF-B-dependent transcription, although less efficiently than GR wt (Kunz et al, 2003).…”

Unbinding of Retinoic Acid from the Retinoic Acid Receptor by Random Expulsion Molecular Dynamics

“…Experiences from previous MD studies of nuclear receptors (20,21) guided our setup of a RAR model for REMD simulations. RAR was solvated in a spherical water droplet to enable complete solvation of surface residues with a limited number of water molecules.…”

A Conserved Protein Motif Is Required for Full Modulatory Activity of Negative Elongation Factor Subunits NELF-A and NELF-B in Modifying Glucocorticoid Receptor-regulated Gene Induction Properties

“…3 Present address: Radiation Oncology, University of Florida, Gainesville, FL 32611. 4 To whom correspondence should be addressed: Bldg. 10, Room 8N-307B, NIDDK/LERB, National Institutes of Health, Bethesda, MD 20892-1772.…”