Overexpression of the NCoA-1 and NCoA-3 PAS-B domains or a CID/AD1 fragment differentially affects transcriptional activation by nuclear receptors. (A) Schematic structure of NCoA-1 and different NCoA protein constructs. Functional and structural domains of NCoA-1 are depicted. The bHLH domain, PAS domain, NID, CID/AD1, AD2 and corresponding LXXLL motifs (black bars) are indicated. Terminal amino acids of each construct are shown. HeLa cells were transfected with expression vectors for the luciferase reporter plasmid pGL2-ERE TK-luc (1.25 µg), the estrogen receptor (12.5 ng) and the LacZ expression plasmid (12.5 ng) along with expression plasmids for the PAS-B domains of NCoA-1 (aa 260–370) or NCoA-3 (aa 265–375) or the NCoA-1 CID/AD1 (aa 804–1032) (each 50, 100 or 200 ng) or the empty expression vector (200 ng; contr.) as indicated. Total amount of DNA was adjusted with the empty expression vector. Transfected cells were grown for at least 3 days without 17β-estradiol (E2) and then treated (black bars) or not treated (unstimulated; white bars) with E2 (10⁻⁷ M) for 16 h. Cell extracts were prepared and luciferase and β-galactosidase activities were determined. Luciferase activities were normalized to the LacZ expression. The average of three independent experiments ± standard deviation is shown. (B) LnCap cells were transduced with lentiviruses encoding the PAS-B domain of NCoA-1 (pVIG-Flag-NCoA-1 aa 260–370), NCoA-3 (pVIG-Flag-NCoA-3 aa 265–375) or the empty vector (pVIG-Flag) alone. The transduction efficiency for the PAS-B domain of NCoA-1 (88–95%), NCoA-3 (32–54%) and the empty vector (92–95%) was confirmed by FACS analysis on the basis of GFP co-expression. Cells were grown for at least 3 days in absence of androgen, followed by treatment (dark grey bars) or no treatment [EtOH (vehicle; ethanol), light grey bars] with dihydrotestosterone (DHT; 100 nM) for 16 h. Total cellular RNA of transduced cells was extracted and transcribed to cDNA. Relative PSA (prostate specific antigen) expression was quantified via quantitative RT-PCR using specific primers. mRNA levels of PSA were normalized against endogenous 18S mRNA. A 12-fold overexpression of the NCoA-1 PAS-B domain and a 2-fold overexpression of the NCoA-3 PAS-B versus endogenous NCoA-1 or NCoA-3, respectively were also determined via quantitative RT-PCR with specific primers. A representative result of three independent transduction of LnCap cells is shown with standard derivation from triplicate RT-PCR measurements.

Models for the recruitment of NCoA proteins. (A) Model for the STAT6-mediated recruitment of NCoA-1. The contact of the PAS-B domain of NCoA-1 to the STAT6 LXXLL motif abolishes the intermolecular interaction of two NCoA-1 proteins. Interaction of p300/CBP with NCoA-1 stabilizes the contact of p300/CBP with STAT6 and enables transcriptional activation. (B) Model for the recruitment of pairs of NCoA proteins to steroid hormone receptor (SR) target gene promoters. The postulated co-recruitment of pairs of NCoA-1 and NCoA-3 is mediated through the PAS-B domain and the CID/AD1.

LXXLL motifs differentially contribute to the interaction of the PAS-B domains and the CID/AD1 of NCoA-1. (A) Sequence alignment of the fragments in the CID/AD1 of NCoA-1 and NCoA-3. LXXLL motifs are printed in bold. The regions of NCoA-1 and NCoA-3 which mediate the interaction with CBP are indicated in boxes (NCoA-1 aa 926–960 and NCoA-3 aa 1045–1091). Terminal amino acids of each construct are shown. Sequences of NCoA-1 and NCoA-3 correspond to the protein ID's AAI11534 and AAC51677, respectively. (B) Fragments of NCoA-1 comprising amino acids 901–970, 901–937 or 938–970 and NCoA-3 comprising amino acids 1022–1092 of the CID/AD1 were in vitro transcribed/translated and [³⁵S]methionine-labeled. The fragments were incubated with GST alone or GST fusion proteins of the PAS-B domains of all three NCoA family members or a fusion protein of the CBP NCoA interacting region (aa 2058–2130) bound to glutathione Sepharose. Precipitated proteins were analyzed by SDS–PAGE and fluorography. 10% of radioactive-labeled material used for interaction assays was analyzed in parallel (Input). (C) Structure of NCoA-1. Different functional domains (grey boxes) and LXXLL motifs (black bars) are indicated. Peptides derived from the CID/AD1 used in competition experiments are shown. The sequences representing LXXLL motif 4 and motif 5 are printed in bold. GST pulldown assays were performed as described in B, with the NCoA PAS-B domains and the [³⁵S]methionine-labeled fragment of the NCoA-1 CID/AD1 in absence or presence of 280, 28 or 2.8 µM of each peptide. (D) Schematic representation of the NCoA-1 CID/AD1 sequence. LXXLL motifs 4 and 5 are printed in bold. Point mutations of leucines to alanines are depicted for motif 4 (MutM4), motif 5 (MutM5) and both motifs (MutM4+5). (E) Experiments were performed as described in B with the radioactive-labeled fragments containing residues 901–970 of wild type or mutant (MutM4, MutM5 or MutM4+5) NCoA-1. (F) Quantification of bound radioactive-labeled fragments of wild type or mutant NCoA-1 CID/AD1 (aa 901–970) to GST and GST fusion proteins. Relative radioactivity (cpm; counts per minute) was determined by normalization against an aliquot representing 10% of the radioactive-labeled material. The average of three independent experiments with standard deviation is shown. (G) Chemical shift perturbation plot on NCoA-1 sequence upon peptides binding. ¹H-¹⁵N HSQC titration data for the addition of LXXLL peptides to the ¹⁵N -NCoA-1 PAS-B domain Δδ values (calculated as described in Materials and Methods) generated upon motif 4 (blue bars), motif 5 (red bars) and STAT6 peptide (green bars) binding are plotted against the NCoA-1 PAS-B domain sequence.

NCoA proteins interact in cells. Whole cell extracts of 293T and LnCap cells were prepared and immunoprecipitation (IP) was performed with an anti-NCoA-1-specific, an anti-NCoA-2-specific, an anti-NCoA-3-specific or an unspecific antibody (IgG). All samples were analyzed by SDS–PAGE and western blotting with specific antibodies against NCoA-1, NCoA-2 or NCoA-3, vice versa to immunoprecipitation. As a control 1% of the cell lysates were analyzed in parallel (Input 1%).

Interaction of NCoA proteins through the PAS-B domains and CID/AD1. (A) Schematic representation of NCoA-1, different NCoA-1 constructs and the STAT6-TAD fragment. bHLH domain, PAS domain, NID [three LXXLL motifs (black bars)], CID/AD1 (two LXXLL motifs) and AD2 (one LXXLL motif) are indicated. The STAT6 peptide comprises the NCoA-1 interacting LXXLL motif (black bar). The terminal amino acids of each construct are shown. The NCoA-1 fragments representing different regions containing LXXLL motifs were in vitro transcribed/translated and [³⁵S]methionine-labeled. A STAT6 fragment which contains the NCoA-1 interacting LXXLL motif was used as a control. After incubation with GST or GST fusion proteins of NCoA-1, NCoA-2 or NCoA-3 PAS-B domains, bound to glutathione Sepharose beads, proteins were eluted and analyzed by SDS–PAGE and fluorography. As an input control 10% of the radioactive-labeled proteins were analyzed in parallel. (B) Experiments were performed with the radioactive-labeled full-length NCoA-1, NCoA-2 and NCoA-3 as described before. (C) 293T cells were transiently transfected with expression vectors encoding either a YFP fusion protein of NCoA-1 aa 213–462, containing the PAS-B domain, or CFP and full-length NCoA-1 (each 3 µg). Cell extracts were prepared and immunoprecipitation (IP) was performed with an anti-GFP antibody, recognizing CFP and YFP. Proteins were analyzed by SDS–PAGE and western blotting with an anti-NCoA-1 antibody. As a control 1% of the cell lysates were analyzed in parallel (Input 1%). (D) The GST pulldown assay was performed with the GST fusion protein comprising the aminoterminal region of NCoA-1 (aa 1–370) and the [³⁵S]methionine-labeled fragments of NCoA-1 containing the aminoterminal region or the CID/AD1 (aa 804–1032).

Overexpression of the NCoA PAS-B domain or competition with the CID/AD1 inhibits contact of NCoA-1 to CBP or the STAT6-TAD. (A) 293T cells were transiently transfected with expression vectors encoding a FLAG-tagged CBP fragment (aa 2058–2130) or the empty FLAG-tag vector (pMX-FLAG) (each 1 µg), a 6× Myc-tagged CID/AD1 fragment of NCoA-1 (aa 804–1032) (0,1 µg) along with a vector containing a YFP fusion protein encoding NCoA-1 aa 213–462 or CFP as a control (each 3 µg). Whole cell extracts were prepared and co-immunoprecipitation was performed with a specific anti-FLAG-tag antibody. Precipitated proteins were analyzed by SDS–PAGE, followed by western blotting with an anti-Myc-tag antibody. Aliquots corresponding to 1% of the used cell lysates were analyzed in parallel (Input 1%). (B) GST or the GST fusion protein of NCoA-1 PAS-B domain (aa 260–370) were bound to glutathione Sepharose beads and incubated with a [³⁵S]methionine-labeled fragment of the STAT6-TAD (aa 792–847) in absence or presence of 45 µM of a purified NCoA-1 CID/AD1 peptide (aa 901–970). After washing and elution, precipitated proteins were analyzed by SDS–PAGE and fluorography. The CID/AD1 fragment was bacterially expressed, digested and purified as described in Materials and Methods. An aliquot representing 10% of the in vitro transcribed/translated fragment was analyzed in parallel (Input). A quarter of each binding reaction was separately analyzed by SDS–PAGE and Coomassie staining in order to prove same amounts of GST or GST fusion proteins. (C) Quantification of bound STAT6-TAD fragment to GST or GST fusion protein of the NCoA-1 PAS-B domain in absence or presence of the CID/AD1 peptide. The relative radioactivity (cpm; counts per minute) was determined by normalization against 10% of the in vitro transcribed/translated protein. The average of three independent experiments with standard deviation is shown.