Ligand-independent coregulator recruitment by the triply activatable OR1/retinoid X receptor-alpha nuclear receptor heterodimer

OR1 is a member of the superfamily of steroid/thyroid hormone nuclear recep tors and recognizes DNA as a heterodimer with the 9-cis-retinoic acid recep tor RXR (retinoid X receptor). The heterodimeric complex has been shown to be transcriptionally activatable by the RXR ligand as well as certain oxyst erols via OR1, but to date uniquely also by heterodimerization itself. Rece nt studies on other members of the superfamily of nuclear receptors have le d to the identification of a number of nuclear receptor-interacting protein s that mediate their regulatory effects on transcription. Here, we address the question of involvement of some of these cofactors in the three modes o f activation by the OR1/RXR alpha complex. We show that in vitro the steroi d receptor coactivator SRC-1 can be recruited by RXR alpha upon addition of its ligand, and to OR1 upon addition of 22(R)-OH-cholesterol, demonstratin g that the latter can act as a direct ligand to OR1. Additionally, heterodi merization is sufficient to recruit SRC-1 to OR1/RXR alpha, indicating SRC- 1 as a molecular mediator of dimerization-induced activation. In transfecti on experiments, coexpression of a nuclear receptor-interacting fragment of SRC-1 abolishes constitutive activation by OR1/RXR alpha, which can be rest ored by overexpression of full-length SRC-1. This constitutes evidence for an in vivo role of SRC-1 in dimerization-induced activation by OR1/RXR alph a. Additionally, we show that the nuclear receptor-interacting protein RIP140 binds in vitro to OR1 and RXR alpha with requirements distinct from those o f SRC-1, and that binding of the two cofactors is competitive. Taken togeth er, our results suggest a complex modulation of differentially induced tran sactivation by OR1/RXR coregulatory molecules.