Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2010) 21 OC5.1

Steroids and thyroid

Familial GC resistance: a novel, naturally occurring mutation which has dominant negative effects on ligand-dependent and -independent GR action

P Trebble1, L Matthews1, J Blaikley1, A Wayte2, G Black1, A Wilton2 & D Ray1

1Department of Medicine, University of Manchester, Manchester, UK; 2Department of Medicine and Clinical Biochemistry, Ysbyty Gwynedd, Bangor, UK.

Glucocorticoids (GCs) are the most potent anti-inflammatory agents known and are used extensively in the treatment of inflammatory disease, however an individuals response to therapy varies greatly. Resistance to GC is often attributed to mutations within the glucocorticoid receptor (GR) through which GC elicit their effects. Here, we present a kindred with GR haploinsufficiency yet no clinical phenotype. PCR and GR sequence profiling of genomic DNA isolated from patient blood reveals a single nucleotide deletion in exon 6, producing a frameshift at residue 612 and introduction of a stop codon at residue 627. This generates 15 novel C-terminal amino acids and results in truncation and loss of a significant portion of the ligand binding domain. In this study we explore the effect of 627GR in isolation, and its effect on GRα. In vitro studies show that 627GR is neither downregulated nor phosphorylated on residue serine211 following treatment with the synthetic GC dexamethasone, two markers of ligand-dependant GR activity. 627GR failed to transactivate a simple reporter (TAT3-Luc) and antagonised the effects of GRα when co-expressed (50% reduction) suggesting dominant negative activity. 627GR alone was unable to repress NRE-Luc and did not antagonise the dexamethasone-mediated repression by GRα. Interestingly 627GR antagonised ligand-independent repression of NRE-Luc by GRα. Fluorescent microscopy using GFP-tagged 627GR revealed that 627GR failed to translocate into the nucleus in the presence of dexamethasone. Since it appears unable to bind ligand and does not translocate into the nucleus to regulate target genes, it is possible that the dominant negative effects of 627GR on GRα may be via heterodimerisation and cytoplasmic sequestration of GRα or non-genomic events initiated in the cytoplasm. The fact that these patients presented with a very mild phenotype, but had dramatically impaired GR function has implications for understanding human GC sensitivity.

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