Endocrine Abstracts

Glucocorticoids (Gcs) act through the ubiquitously expressed glucocorticoid receptor (GR). Transcriptional activation by the GR involves interaction with co-modulator proteins, the expression level of which can mediate Gc sensitivity. The aim of this study was to identify additional GR interacting proteins that may influence Gc sensitivity.

Using a yeast two-hybrid screen we identified a novel GR interacting protein, R59, which showed an enhanced interaction with the GR in the presence of GR antagonist, RU486. Database analysis identified 100% identity of this sequence with a sequence on human chromosome 17. Bioinformatics analysis identified a 4-exon gene, with an open reading frame identical to the identified cDNA. This predicted a spliced transcript 3 kb in length and a 121 amino acid sequence encoding a 14 kDa protein, conserved in human, mouse and rat genomes. Northern blot analysis identified a prominent transcript of 3 kb with high-level expression in human placenta, heart and muscle. Western blot analysis using an antibody specific to R59 identified a 14 kDa protein in a non-small cell lung cancer cell line A549, 2 small cell lung cancer cell lines CORL24 and DMS79, HEK, CEM-7A and Cos7 cells. No expression was found in normal lung. Immunofluorescence revealed mainly cytoplasmic expression with clustering around the nuclear membrane and movement to the nucleus with GR-EYFP upon dexamethasone treatment. In a GST pull down assay, R59 interacted constitutively with full length GR with no additional effect of ligand binding. Over-expression of R59 in A549 cells inhibited Gc transactivation of a simple reporter gene (TAT₃-luc) but modestly enhanced Gc transactivation of the same reporter gene in R59-negative HepG2 cells. R59 was downregulated in A549 cells by PMA, staurosporine, triclostatin A and by dexamethasone dose-dependently.

We have identified a novel GR interacting protein that may play an important role in the mechanism of Gc-sensitivity in certain disease states.