YEAST HORMONE RESPONSE ELEMENT ASSAYS DETECT AND CHARACTERIZE GRIP1 COACTIVATOR-DEPENDENT ACTIVATION OF TRANSCRIPTION BY THYROID AND RETINOID NUCLEAR RECEPTORS

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a mem ber of the ERAP160 family of nuclear receptor (NR) coactivators (inclu ding SRC-1 and TIF2) which function as bridging proteins between ligan d-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivat ors bind to several NRs, studies overexpressing these coactivators wit h these NRs in mammalian cells have not uniformly observed a correspon ding enhancement of ligand-dependent transactivation. Here, we show th at GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionall y, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein marke dly increased the ability of these full-length class II NRs to transac tivate beta-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, th yroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Alth ough GRIP1 exerted no significant effect on NR homodimers in the absen ce of their cognate ligands, it increased the transactivation of unlig anded NR heterodimers. Whether GRIP1 increased ligand-dependent transa ctivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compare d with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilita ted both the detection of putative mammalian NR coactivator function a nd the elucidation of their mechanisms of transactivational enhancemen t.