Endocrine Abstracts

Glucocorticoid receptor (GR) splicing may influence glucocorticoid sensitivity. We aimed to identify GR isoforms in the lung by using immunohistochemistry, immunoblotting, co-immunoprecipitation western and RT-PCR approaches.
The distribution of GR protein in rat lung was different when we used an N terminal antibody (M20) compared to a C terminal antibody (GRalpha). M20 detected diffuse expression in all cell types and higher expression in the epithelium. In contrast GRalpha only picked out the airway epithelial cells, airway smooth muscle cells and blood vessels. These findings were confirmed by immunoadsorption.
Immunoblotting studies with M20 showed a single 98kD band, which was abolished by immunoadsorption. We immunoprecipitated samples using M20 then immunoblotted with either M20 or GRalpha and found a single band at 98kD under both conditions, corresponding to the band identified by direct immunoblotting. However, as the immunohistochemistry identified different GR species we silver stained the immunoprecipitate and identified three bands with molecular mass close to 98kD, likely to represent alternate GR isoforms.
We also analysed GR protein expression in the human lung cell line A549. Using the discriminatory IP-western approach two proteins were detected by M20, one at 98kD and the other at 85kD. GR identity of both bands was confirmed by immunoadsorption. Both 98 and 85kD proteins were repressed by dexamethasone treatment. Only the 98kD protein was also detected using GRalpha, confirming its identity as full-length GRalpha. There was no GRbeta mRNA detectable in A549 cells using RT-PCR. As the 85kD GR species is larger than the reported GRdelta form (74kD), and smaller than GRbeta (94kD) we propose that it is a novel GR isoform.
In summary we present evidence of novel GR variants in human and rat lung. We propose that these influence glucocorticoid sensitivity and we plan to characterise their regulation and function in further studies.