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Glucocorticoids are potent anti-inflammatory agents, acting through the glucocorticoid receptor (GR) to regulate target gene transcription. However, GR may also exert acute effects, including activation of signaling kinases such as c-Src and protein kinase B, possibly via the scaffold protein, modulator of nongenomic action of the estrogen receptor (MNAR). MNAR inhibited GR transactivation in A549 cells, but in HEK293 cells there was a ligand concentration-dependent biphasic effect. Transactivation driven by low ligand concentrations was inhibited by MNAR expression, whereas higher ligand concentrations were potentiating. Further analysis revealed that MNAR inhibited transactivation by the ligand-independent activation function (AF)1 but potentiated the COOH-terminal AF2 domain. The effect of MNAR was independent of c-Src activity, demonstrated by inhibitors and c-Src knockdown studies. In support of the role of MNAR in modulating GR transactivation, coimmunoprecipitation studies showed interaction between MNAR and GR in the nucleus but not the cytoplasm. Furthermore, MNAR and c-Src were also found to physically interact in the nucleus. Immunofluorescence studies showed MNAR to be predominantly a nuclear protein, with significant colocalization with GR. Deletion studies revealed that MNAR 884-1130 was coimmunoprecipitated with GR, and furthermore this fragment inhibited GR transactivation function when overexpressed. In addition, MNAR 1-400, which contains multiple LxxLL motifs, also inhibited GR transactivation. Taken together, MNAR interacts with GR in the nucleus but not cytoplasm and regulates GR transactivation in a complex manner depending on cell type. MNAR is capable of regulating both AF1 and AF2 functions of the GR independently. MNAR expression is likely to mediate important cell variation in glucocorticoid responsiveness, in a c-Src-independent mechanism.

Original publication

DOI

10.1152/ajpendo.90429.2008

Type

Journal article

Journal

Am J Physiol Endocrinol Metab

Publication Date

11/2008

Volume

295

Pages

E1047 - E1055

Keywords

Binding Sites, Cell Line, Cell Line, Tumor, Cell Nucleus, Co-Repressor Proteins, Cytoplasm, Dexamethasone, Humans, Immunoprecipitation, Phosphorylation, Protein Binding, Protein Kinase Inhibitors, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, RNA Interference, Receptors, Glucocorticoid, Sequence Deletion, Trans-Activators, Transcription Factors, Transcriptional Activation, Transfection, src-Family Kinases