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 OR 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 OR transactivation, coimmunoprecipitation studies showed interaction between MNAR and OR 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 OR. Deletion studies revealed that MNAR 884-1130 was coimmunoprecipitated with OR, and furthermore this fragment inhibited OR transactivation function when overexpressed. In addition, MNAR 1-400, which contains multiple LxxLL motifs, also inhibited OR transactivation. Taken together, MNAR interacts with OR in the nucleus but not cytoplasm and regulates OR transactivation in a complex manner depending on cell type. MNAR is capable of regulating both AFI and AF2 functions of the OR independently. MNAR expression is likely to mediate important cell variation in glucocorticoid responsiveness, in a c-Src-independent mechanism. [ABSTRACT FROM AUTHOR]