Identification and localization of steroid-binding and nonsteroid-binding forms of the glucocorticoid receptor in the mouse P1798 lymphosarcoma

Abstract

Glucocorticoid receptors (GCRs) were characterized in sublines of the mouse P1798 lymphosarcoma that are sensitive (S) or resistant (R) to glucocorticoid-mediated apoptosis. Previous work had identified two steroid-binding GCRs in S and R cells: a 97 kDa wild-type GCR in S cells (WT-GCR), and a 45 kDa truncated GCR in R cells (TR-GCR). A third GCR, a 97 kDa nonsteroid-binding GCR (NSB-GCR), was also identified in R cells. Using subcellular fractionation and Western blotting, we now show that in contrast to the WT-GCR which is localized in both the cytoplasm and nucleus of S cells, the NSB-GCR is localized predominantly in R cell nuclei. Moreover, gel filtration chromatography revealed that treatment with 400 mM NaCl and heat did not significantly alter the Stokes radius of the NSB-GCR suggesting that this receptor is not present in a heterooligomeric complex with other proteins. The TR-GCR was localized predominantly in the soluble cytoplasmic fraction but also in the crude membrane fractions of R cell nuclei, suggesting that this receptor is tightly associated with nuclear structures. It was not detected in the soluble nuclear fraction. Unexpectedly, a 45 kDa nonsteroid-binding immunoreactive protein was detected in crude membrane fractions of S cells. These studies describe a complex GCR system in the P1798 lymphosarcoma that necessitates a further consideration of glucocorticoid signaling in S and R cells.