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/retinoid receptor superfamily are ligand-inducible transcription factors. These receptors modulate transcription of specific cellular genes, either positively or negatively, by interacting with specific hormone response elements located near the target promoters. Recent studies indicated that the hormone- occupied, DNA-bound receptor acts in concert with a cellular coregulatory factor, termed coactivator, and the basal transcription machinery to mediate gene activation. Consistent with this scenario, a number of nuclear proteins with potential coactivator function have been isolated. In the present study, we demonstrate that steroid receptor coactivator-1 (SRC-1), a recently isolated candidate coactivator, functions as a positive regulator of the thyroid hormone receptor (TR)-mediated transactivation pathway. In transient transfection experiments, coexpression of SRC-1 significantly enhanced ligand-dependent transactivation of a thyroid hormone response element (TRE)-linked promoter by human TRbeta. Our studies revealed that deletion of six amino acids (451-456) in the extreme COOH-terminal region of TRbeta resulted in a receptor that retained the ability to bind T3 but failed to be stimulated by SRC-1. These six amino acids are part of an amphipathic 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 binding studies showed that SRC-1 interacted with a ligand binding domain peptide (145-456) of TRbeta in a T3-dependent manner, whereas it failed to interact with a mutant ligand binding domain lacking the amino acids (451-456). We demonstrated that a synthetic peptide containing the COOH-terminal amino acids (437-456) of TRbeta efficiently blocked 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 TRbeta is critical for interaction with SRC-1 and thereby plays a central role in coactivator-mediated transactivation. We further observed that a heterodimer of TRbeta and retinoid X receptor-alpha (RXR alpha), either in solution or bound to a DR+4 TRE, recruited SRC-1 in a T3-dependent manner. The AF-2 of TR was clearly involved in this process because a TR-RXR heterodimer containing a mutant TRbeta (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 raise the interesting possibility that a RXR-specific ligand may modulate T3-mediated signaling by inducing additional interactions between TRE-bound TR-RXR heterodimer and the coactivator.
Jeyakumar
M
M
The Population Council and The Rockefeller University, New York, New York 10021, USA.
Tanen
M R
MR
Bagchi
M K
MK
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D019281:Dimerization; D005786:Gene Expression Regulation; D051548:Histone Acetyltransferases; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D056920:Nuclear Receptor Coactivator 1; D010455:Peptides / Q000138:chemical synthesis; D011817:Rabbits; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D011988:Receptors, Thyroid Hormone / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
D008024:Ligands; D010455:Peptides; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1