Specific Structural Motifs Determine TRAP220 Interactions with Nuclear Hormone Receptors

ABSTRACT The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D3 receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.

[1]  J. Lehmann,et al.  Formation of retinoid X receptor homodimers leads to repression of T3 response: hormonal cross talk by ligand-induced squelching , 1993, Molecular and cellular biology.

[2]  M. Rao,et al.  Isolation and Characterization of PBP, a Protein That Interacts with Peroxisome Proliferator-activated Receptor* , 1997, The Journal of Biological Chemistry.

[3]  Robert Tjian,et al.  The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1 , 1999, Nature.

[4]  R. Roeder,et al.  Expression and purification of general transcription factors by FLAG epitope-tagging and peptide elution. , 1993, Peptide research.

[5]  K. Ichikawa,et al.  Ligand-dependent Heterodimerization of Thyroid Hormone Receptor and Retinoid X Receptor* , 1997, The Journal of Biological Chemistry.

[6]  B. O’Malley,et al.  Molecular mechanisms of action of steroid/thyroid receptor superfamily members. , 1994, Annual review of biochemistry.

[7]  D. Reinberg,et al.  NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription. , 1998, Molecular cell.

[8]  P. Hallenbeck,et al.  Divergent effects of 9-cis-retinoic acid receptor on positive and negative thyroid hormone receptor-dependent gene expression. , 1993, The Journal of biological chemistry.

[9]  E. Kalkhoven,et al.  AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors , 1997, Molecular and cellular biology.

[10]  David M. Heery,et al.  A signature motif in transcriptional co-activators mediates binding to nuclear receptors , 1997, Nature.

[11]  R. Evans,et al.  The RXR heterodimers and orphan receptors , 1995, Cell.

[12]  A. Wolffe,et al.  Determinants of chromatin disruption and transcriptional regulation instigated by the thyroid hormone receptor: hormone‐regulated chromatin disruption is not sufficient for transcriptional activation , 1997, The EMBO journal.

[13]  J. Thomsen,et al.  Competition between Thyroid Hormone Receptor-associated Protein (TRAP) 220 and Transcriptional Intermediary Factor (TIF) 2 for Binding to Nuclear Receptors , 1999, The Journal of Biological Chemistry.

[14]  N. Webster,et al.  The human estrogen receptor has two independent nonacidic transcriptional activation functions , 1989, Cell.

[15]  J. Lees,et al.  Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. , 1992, The EMBO journal.

[16]  David A. Agard,et al.  The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen , 1998, Cell.

[17]  M. Lazar,et al.  The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes , 2000, Molecular and Cellular Biology.

[18]  D. Barettino,et al.  Characterization of the ligand‐dependent transactivation domain of thyroid hormone receptor. , 1994, The EMBO journal.

[19]  B. O’Malley,et al.  The tau 4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing , 1995, Molecular and cellular biology.

[20]  Thorsten Heinzel,et al.  A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors , 1996, Cell.

[21]  H. Gronemeyer,et al.  The nuclear receptor ligand-binding domain: structure and function. , 1998, Current opinion in cell biology.

[22]  C. Carlberg,et al.  Two nuclear signalling pathways for vitamin D , 1993, Nature.

[23]  Jacek Ostrowski,et al.  Two distinct actions of retinoid-receptor ligands , 1996, Nature.

[24]  Daniel Metzger,et al.  Activation of the Estrogen Receptor Through Phosphorylation by Mitogen-Activated Protein Kinase , 1995, Science.

[25]  F. Claessens,et al.  The Androgen Receptor Amino-Terminal Domain Plays a Key Role in p160 Coactivator-Stimulated Gene Transcription , 1999, Molecular and Cellular Biology.

[26]  J. Fondell,et al.  Identification of mouse TRAP100: a transcriptional coregulatory factor for thyroid hormone and vitamin D receptors. , 1999, Molecular endocrinology.

[27]  R. Evans,et al.  Regulation of Hormone-Induced Histone Hyperacetylation and Gene Activation via Acetylation of an Acetylase , 1999, Cell.

[28]  A. Mahfoudi,et al.  Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Parker,et al.  Molecular Determinants of the Estrogen Receptor-Coactivator Interface , 1999, Molecular and Cellular Biology.

[30]  R. Roeder,et al.  The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Roeder,et al.  Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression. , 1993, Genes & development.

[32]  M. Karin,et al.  A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor) , 1993, Molecular and cellular biology.

[33]  C. Glass,et al.  Co-activators and co-repressors in the integration of transcriptional responses. , 1998, Current opinion in cell biology.

[34]  M. Parker,et al.  Steroid and related receptors. , 1993, Current opinion in cell biology.

[35]  R. Roeder,et al.  Unliganded thyroid hormone receptor alpha can target TATA-binding protein for transcriptional repression , 1996, Molecular and cellular biology.

[36]  E. Rosen,et al.  Ligand-dependent synergy of thyroid hormone and retinoid X receptors. , 1992, The Journal of biological chemistry.

[37]  C. Glass,et al.  Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. , 1998, Genes & development.

[38]  I B Dawid,et al.  Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression , 1997, Molecular and cellular biology.

[39]  K. Umesono,et al.  Unique response pathways are established by allosteric interactions among nuclear hormone receptors , 1995, Cell.

[40]  C. Glass,et al.  Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators , 1998, Nature.

[41]  P. Chambon,et al.  Activation function 2 (AF‐2) of retinoic acid receptor and 9‐cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF‐2 activity. , 1994, The EMBO journal.

[42]  R. Brent,et al.  Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. , 1995, Molecular endocrinology.

[43]  K. Umesono,et al.  Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors , 1992, Nature.

[44]  J. Qin,et al.  A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. , 1999, Molecular cell.

[45]  K. Yamamoto,et al.  An Additional Region of Coactivator GRIP1 Required for Interaction with the Hormone-binding Domains of a Subset of Nuclear Receptors* , 1999, The Journal of Biological Chemistry.

[46]  H. Erdjument-Bromage,et al.  A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system. , 1998, Genes & development.

[47]  M. Stallcup,et al.  Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. , 1998, Molecular Endocrinology.

[48]  C. Glass,et al.  Regulation of retinoid signalling by receptor polarity and allosteric control of ligand binding , 1994, Nature.

[49]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[50]  S. Minucci,et al.  The histone acetylase PCAF is a nuclear receptor coactivator. , 1998, Genes & development.

[51]  R. Roeder,et al.  Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[52]  E. Kalkhoven,et al.  Isoforms of steroid receptor co‐activator 1 differ in their ability to potentiate transcription by the oestrogen receptor , 1998, The EMBO journal.

[53]  G. Coetzee,et al.  Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins , 1999, Molecular and Cellular Biology.

[54]  M. Montminy,et al.  Role of CBP/P300 in nuclear receptor signalling , 1996, Nature.

[55]  H. Gronemeyer,et al.  The coactivator TIF2 contains three nuclear receptor‐binding motifs and mediates transactivation through CBP binding‐dependent and ‐independent pathways , 1998, The EMBO journal.

[56]  R. Frade,et al.  Identification of RB18A, a 205 kDa new p53 regulatory protein which shares antigenic and functional properties with p53 , 1997, Oncogene.

[57]  M. Lazar,et al.  A Novel Role for Helix 12 of Retinoid X Receptor in Regulating Repression , 1999, Molecular and Cellular Biology.

[58]  P. Chambon,et al.  Promoter context- and response element-dependent specificity of the transcriptional activation and modulating functions of retinoic acid receptors , 1992, Cell.

[59]  F. Jeanmougin,et al.  A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors. , 1996, The EMBO journal.

[60]  C. Allis,et al.  Steroid receptor coactivator-1 is a histone acetyltransferase , 1997, Nature.

[61]  R. Evans,et al.  Nuclear Receptor Coactivator ACTR Is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300 , 1997, Cell.

[62]  T. Willson,et al.  Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-γ , 1998, Nature.

[63]  R. Roeder,et al.  Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[64]  R. Lanz,et al.  Nuclear receptor coregulators: cellular and molecular biology. , 1999, Endocrine reviews.

[65]  R J Fletterick,et al.  Structure and specificity of nuclear receptor-coactivator interactions. , 1998, Genes & development.

[66]  Christopher K. Glass,et al.  The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function , 1997, Nature.

[67]  J. Direnzo,et al.  p300 is a component of an estrogen receptor coactivator complex. , 1996, Proceedings of the National Academy of Sciences of the United States of America.