An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

Abstract

The NH 2 -terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH 2 -terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH 2 -terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH 2 -terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH 2 -terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH 2 -terminal domains and other intrinsically unstructured transcriptional regulatory proteins.Steroid receptors depend on multiple domains for their function as ligand-dependent transcriptional activators. The DNAand ligand-binding domains have been studied extensively and have a high degree of structural and functional conservation. In contrast, the largely unstructured NH 2 -terminal domains (1-3) are highly variable in size and sequence, and the molecular mechanisms that contribute to transactivation are not well understood. The size of the NH 2 -terminal region of steroid receptors increases with evolutionary expansion (4, 5), from estrogen receptor-␣ (185 amino acid residues) to the glucocorticoid receptor (GR 1 ; 420 residues), androgen receptor (AR; 558 residues), progesterone receptor (B form; 566 residues), and mineralocorticoid receptor (602 residues). In contrast, transcription factors such as p53, NF-B, and VP16 typically have transcriptional activation domains of Ͻ100 residues.

A gene signature identified using a mouse model of androgen receptor‐dependent prostate cancer predicts biochemical relapse in human disease

“…AR-E231G exhibits increased responsiveness to androgens [testosterone and 5a-dihydrotestosterone (DHT)] and estrogens (estradiol) in the presence of the co-activators, ARA70 and ARA160, 13 and decreased responsiveness to the co-repressor, CHIP. 19 Using enforced overexpression of wtAR, AR-T857A (described earlier), or AR-E231G specifically in the prostate of mice, we previously demonstrated the oncogenic capacity of the AR-E231G variant. 8 Although overexpression of wtAR or AR-T857A had no discernable phenotype, mice expressing AR-E231G recapitulated all stages of disease, developing prostatic intraepithelial neoplasia (PIN)-like lesions at 12 weeks of age and advanced prostate cancer with lung metastases by 50 weeks.…”

CHIP Overexpression Reduces Mutant Androgen Receptor Protein and Ameliorates Phenotypes of the Spinal and Bulbar Muscular Atrophy Transgenic Mouse Model

“…Wild-type AR is one of the CHIP substrates (Cardozo et al, 2003;He et al, 2004). CHIP also interacts with misfolded proteins trapped by molecular chaperones and degrades them, thus acting as a "quality control" E3 (Cyr et al, 2002;Murata et al, 2003).…”

Probing the Functional Link between Androgen Receptor Coactivator and Ligand-binding Sites in Prostate Cancer and Androgen Insensitivity

“…Other vectors used were as follows: VP-AR-(1-660) (11,14); transcriptional intermediary factor 2 (TIF2)-AR fusion protein TIF2(LXXLL) 3 AR-(172-919) (18); pSG5-TIF2 and VP-TIF2-(624 -1287) (VP-TIF2.1) (23); wild-type and mutant pcDNA3-AR-(624 -919) (10,14); and 5XGAL4Luc3 (16). GST-SRC1-IV (GST-SRC1-(1139 -1441)) was created by digesting GAL-SRC1-(1139 -1441) with XbaI (blunt-ended) and BamHI, and the fragment was subcloned into pGEX-3X digested with EcoRI (blunt-ended) and BamHI.…”

“…Like other steroid receptors, the androgen receptor (AR) 3 mediates transcriptional activation primarily through two domains. Activation function 1 (AF1) in the NH 2 -terminal region is the major transactivation domain (2) but is not well conserved across species (3).…”

“…Like other steroid receptors, the androgen receptor (AR) 3 mediates transcriptional activation primarily through two domains. Activation function 1 (AF1) in the NH 2 -terminal region is the major transactivation domain (2) but is not well conserved across species (3). Activation function 2 (AF2) in the ligand binding domain is a highly conserved hydrophobic surface that is stabilized by agonist binding and required for SRC/p160 coactivator recruitment.…”