FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor*

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH2-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted α-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH2- and carboxyl-terminal interaction. FXXLF in the AR NH2-terminal domain with the sequence23FQNLF27 mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH2-terminal WXXLF sequence 433WHTLF437 interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation.

[1]  William Bourguet,et al.  A canonical structure for the ligand-binding domain of nuclear receptors , 1996, Nature Structural Biology.

[2]  T. Ikonen,et al.  Interaction between the Amino- and Carboxyl-terminal Regions of the Rat Androgen Receptor Modulates Transcriptional Activity and Is Influenced by Nuclear Receptor Coactivators* , 1997, The Journal of Biological Chemistry.

[3]  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.

[4]  H. Gronemeyer,et al.  Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain* , 1999, The Journal of Biological Chemistry.

[5]  F. Claessens,et al.  The AF1 and AF2 Domains of the Androgen Receptor Interact with Distinct Regions of SRC1 , 1999, Molecular and Cellular Biology.

[6]  C. Glass,et al.  Nuclear receptor coactivators. , 1997, Current opinion in cell biology.

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

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

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

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

[11]  F. Ghadessy,et al.  Oligospermic infertility associated with an androgen receptor mutation that disrupts interdomain and coactivator (TIF2) interactions. , 1999, The Journal of clinical investigation.

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

[13]  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.

[14]  R. Schüle,et al.  FHL2, a novel tissue‐specific coactivator of the androgen receptor , 2000, The EMBO journal.

[15]  M. Stallcup,et al.  Enhancement of Estrogen Receptor Transcriptional Activity by the Coactivator GRIP-1 Highlights the Role of Activation Function 2 in Determining Estrogen Receptor Pharmacology* , 1998, The Journal of Biological Chemistry.

[16]  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.

[17]  C. Glass,et al.  Molecular determinants of nuclear receptor-corepressor interaction. , 1999, Genes & development.

[18]  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.

[19]  F. S. French,et al.  Transcriptional activation and nuclear targeting signals of the human androgen receptor. , 1991, The Journal of biological chemistry.

[20]  L. Freedman.  Increasing the Complexity of Coactivation in Nuclear Receptor Signaling , 1999, Cell.

[21]  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.

[22]  Paul Tempst,et al.  Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex , 1999, Nature.

[23]  J. A. Kemppainen,et al.  Intermolecular NH2-/Carboxyl-terminal Interactions in Androgen Receptor Dimerization Revealed by Mutations That Cause Androgen Insensitivity* , 1998, The Journal of Biological Chemistry.

[24]  S. Yamashita,et al.  Two Distinct Isoforms of cDNA Encoding Rainbow Trout Androgen Receptors* , 1999, The Journal of Biological Chemistry.

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

[26]  William Bourguet,et al.  Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-α , 1995, Nature.

[27]  Michael R. Green,et al.  Transcription activation by the adenovirus E1a protein , 1989, Nature.

[28]  Zbigniew Dauter,et al.  Molecular basis of agonism and antagonism in the oestrogen receptor , 1997, Nature.

[29]  D. Piomelli,et al.  Brain cannabinoids in chocolate , 1996, Nature.

[30]  B. Katzenellenbogen,et al.  Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Evans,et al.  Mechanism of corepressor binding and release from nuclear hormone receptors. , 1999, Genes & development.

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

[33]  P. Meltzer,et al.  AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. , 1997, Science.

[34]  R. Tjian,et al.  Composite co-activator ARC mediates chromatin-directed transcriptional activation , 1999, Nature.

[35]  E. Wilson,et al.  A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences. , 1994, The Journal of biological chemistry.

[36]  P. Chambon,et al.  TIF2, a 160 kDa transcriptional mediator for the ligand‐dependent activation function AF‐2 of nuclear receptors. , 1996, The EMBO journal.

[37]  E. Wilson,et al.  Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains. , 1993, The Journal of biological chemistry.

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

[39]  D. Fowlkes,et al.  Dissection of the LXXLL Nuclear Receptor-Coactivator Interaction Motif Using Combinatorial Peptide Libraries: Discovery of Peptide Antagonists of Estrogen Receptors α and β , 1999, Molecular and Cellular Biology.

[40]  R J Fletterick,et al.  Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. , 1998, Science.

[41]  J. Leers,et al.  Mechanistic Principles in NR Box-Dependent Interaction between Nuclear Hormone Receptors and the Coactivator TIF2 , 1998, Molecular and Cellular Biology.

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

[43]  M. Lazar,et al.  Interdomain communication regulating ligand binding by PPAR-γ , 1998, Nature.

[44]  E. Langley,et al.  Evidence for an Anti-parallel Orientation of the Ligand-activated Human Androgen Receptor Dimer (*) , 1995, The Journal of Biological Chemistry.

[45]  D. Robins,et al.  Multiple Receptor Domains Interact to Permit, or Restrict, Androgen-specific Gene Activation* , 1998, The Journal of Biological Chemistry.

[46]  C. Glass,et al.  The coregulator exchange in transcriptional functions of nuclear receptors. , 2000, Genes & development.

[47]  E. Wilson,et al.  Evolution of the Primate Androgen Receptor: A Structural Basis for Disease , 1998, Journal of Molecular Evolution.

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

[49]  B. Katzenellenbogen,et al.  Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

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

[51]  M. Lazar,et al.  The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors , 1999, Nature.