Jon A. Kemppainen

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.

Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain

Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH2- and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH2-/carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activityin vivo. The androgen receptor recruits p160 coactivators through its NH2-terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation.

Intermolecular NH2-/carboxyl-terminal interactions in androgen receptor dimerization revealed by mutations that cause androgen insensitivity

Structural alignment of the human androgen receptor dimer was investigated by introducing steroid binding domain mutations that cause partial or complete androgen insensitivity into fusion proteins containing the full-length androgen receptor or the steroid binding domain. Most of the mutants had unchanged apparent equilibrium androgen binding affinity and increased dissociation rates of [3H]methyltrienolone and required increased dihydrotestosterone concentrations for transcriptional activation. In a 2-hybrid protein interaction assay in mammalian cells, the steroid binding domain interacts with an NH2-terminal-DNA binding domain fragment and with the full-length androgen receptor at physiological androgen concentrations in a dose-dependent manner. However, mutations at Val-889 and Arg-752 disrupt the NH2-/carboxyl-terminal interaction when introduced into the steroid binding domain fragment but not when present in the full-length androgen receptor. The N-C bimolecular interaction reduces the dissociation rate of bound androgen and slows the degradation rate of the carboxyl-terminal steroid binding domain fragment. The results suggest that steroid binding domain residues Val-889 and Arg-752 are critical to the NH2-/carboxyl-terminal interaction and that an intermolecular N-C interaction occurs during receptor dimerization that results in an antiparallel arrangement of androgen receptor monomers.

Evolution of the primate androgen receptor: a structural basis for disease

Abstract. Androgen effects mediated by the androgen receptor (AR) are essential for male reproductive development and virilization. Comparison of AR DNA coding sequence from five primate species, Homo sapiens (human), Pan troglodytes (chimpanzee), Papio hamadryas (baboon), Macaca fascicularis (macaque), and Eulemur fulvus collaris (collared brown lemur), supports their phylogeny with complete conservation of the DNA and steroid binding domain protein sequence. A linear increase in trinucleotide repeat expansion of homologous CAG and GGC sequences occurs in the NH2-terminal transcriptional activation region and is proportional to the time of species divergence. A serine phosphate/glutamine repeat interaction is observed where increasing CAG repeat length is associated with an increased rate of serine 94 phosphorylation. Disparity in the calculated and apparent molecular weight with CAG repeat expansion of an AR NH2-terminal fragment suggests self-aggregation with increasing glutamine repeat length into the pathological range. These results suggest that a CAG/glutamine repeat expanded during divergence of the higher primate species, which may have a direct effect on AR structure and support a common pathway in CAG trigenic diseases in the pathophysiology of neurodegeneration observed in X-linked spinal bulbar and muscular atrophy.