Abstract
Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function.
Highlights
From the ‡Institute of Immunology, Centre de Recherche Public de la Sante /Laboratoire National de Sante, 20A rue Auguste Lumiere, L-1950 Luxembourg, Grand-Duchy of Luxembourg; §Department of Immunology, Research Institute of Psychobiology, University of Trier, D-54290 Trier, Germany
Glucocorticoids exert their therapeutic effects by activating the cytosolic glucocorticoid receptor (GR) leading to classical genomic effects
We report the first study on proteomic effects induced by selective activation of the membrane-bound GR (mGR) only
Summary
The origin and the function of this GR isoform were further investigated in the S-49 mouse T-lymphoma cell line [13,14,15,16,17,18]. To date liposome-based fluorescence amplification techniques have been used [26], allowing the detection of as few as 50 receptor molecules per cell By applying this method, Bartholome et al confirmed the presence of the mGR on CCRF-CEM cells and demonstrated that the mGR is physiologically present in monocytes and B-cells from healthy donors, while circulating T-lymphocytes were consistently negative [22]. We present the first proteomic study of the effects of the mGR selectively activated by BSA-conjugated cortisol in a human lymphoma cell line. We visualized the mGR and its association with Cav-1 using the highly sensitive in situ Proximity Ligation Assay (PLA) [37] This membrane protein seems to modulate Cort-BSA effects, but is not necessary for activity
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