ANALYSIS OF THE FUNCTIONAL-ROLE OF STEROID-RECEPTOR COACTIVATOR-1 IN LIGAND-INDUCED TRANSACTIVATION BY THYROID-HORMONE RECEPTOR

The nuclear hormone receptors belonging to the steroid/thyroid/retinoi d receptor superfamily are ligand-inducible transcription factors. The se receptors modulate transcription of specific cellular genes, either positively or negatively, by interacting with specific hormone respon se elements located near the target promoters. Recent studies indicate d that the hormone- occupied, DNA-bound receptor acts in concert with a cellular coregulatory factor, termed coactivator, and the basal tran scription machinery to mediate gene activation. Consistent with this s cenario, a number of nuclear proteins with potential coactivator funct ion have been isolated. In the present study, we demonstrate that ster oid receptor coactivator-l (SRC-1), a recently isolated candidate coac tivator, functions as a positive regulator of the thyroid hormone rece ptor (TR)-mediated transactivation pathway. In transient transfection experiments, coexpression of SRC-1 significantly enhanced ligand-depen dent transactivation of a thyroid hormone response element (TRE)-linke d promoter by human TR beta. Our studies revealed that deletion of six amino acids (451-456) in the extreme COOH-terminal region of TR beta resulted in a receptor that retained the ability to bind T-3 but faile d to be stimulated by SRC-1. These six amino acids are part of an amph ipathic helix that is highly conserved among nuclear hormone receptors and contains the core domain of the ligand-dependent transactivation function, AF-2. In agreement with this observation, in vitro protein b inding studies showed that SRC-I interacted with a ligand binding doma in peptide (145-456) of TR beta in a T-3-dependent manner, whereas it failed to interact with a mutant ligand binding domain lacking the ami no acids (451-456). We demonstrated that a synthetic peptide containin g the COOH-terminal amino acids (437-456) of TR beta efficiently block ed the ligand-induced binding of SRC-1 to the receptor; These results suggest that the conserved amphipathic helix that constitutes the AF-2 core domain of TR beta is critical for interaction with SRC-1 and the reby plays a central role in coactivator-mediated transactivation. We further observed that a heterodimer of TR beta and retinoid X receptor -alpha (RXR alpha), either in solution or bound to a DR+4 TRE, recruit ed SRC-1 in a T-3-dependent manner. The AF-2 of TR was clearly involve d in this process because a TR-RXR heterodimer containing a mutant TR beta (1-450) with impaired AF-2 failed to bind to SRC-1. Surprisingly, the RXR-specific ligand 9-cis-retinoic acid induced binding of SRC-1 to the RXR component of the TRE-bound heterodimer. This novel finding suggests that RXR, as a heterodimeric partner of TR, has the potential to play an active role in transcriptional regulation. Our results rai se the interesting possibility that a RXR-specific ligand may modulate T-3-mediated signaling by inducing additional interactions between TR E-bound TR-RXR heterodimer and the coactivator.