Core LXXLL Motif Sequences in CREB-binding Protein, SRC1, and RIP140 Define Affinity and Selectivity for Steroid and Retinoid Receptors*

David M. HeeryDagger, Susan Hoare§, Sagair Hussain, Malcolm G. Parker§, and Hilary Sheppard

From the Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH and § Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received for publication, October 16, 2000, and in revised form, November 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An alpha -helical motif containing the sequence LXXLL is required for the ligand-dependent binding of transcriptional co-activators to nuclear receptors. By using a peptide inhibition assay, we have defined the minimal "core" LXXLL motif as an 8-amino acid sequence spanning positions -2 to +6 relative to the primary conserved leucine residue. In yeast two-hybrid assays, core LXXLL motif sequences derived from steroid receptor co-activator (SRC1), the 140-kDa receptor interacting protein (RIP140), and CREB-binding protein (CBP) displayed differences in selectivity and affinity for nuclear receptor ligand binding domains. Although core LXXLL motifs from SRC1 and RIP140 mediated strong interactions with steroid and retinoid receptors, three LXXLL motifs present in the global co-activator CBP were found to have very weak affinity for these proteins. Core motifs with high affinity for steroid and retinoid receptors were generally found to contain a hydrophobic residue at position -1 relative to the first conserved leucine and a nonhydrophobic residue at position +2. Our results indicate that variant residues in LXXLL core motifs influence the affinity and selectivity of co-activators for nuclear receptors.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ligand-dependent gene expression mediated by nuclear receptors involves the recruitment of transcriptional co-activators to the ligand binding domain (LBD).1 The LBD is structurally conserved among the NR family and consists of between 10 and 12 alpha -helices folded in a globular domain (1). A central hydrophobic pocket accommodates the cognate ligand, which upon binding induces a conformational change in the LBD, exposing a co-activator docking site on the LBD surface. Sequence conservation, mutational analyses, and crystal structures have indicated that the co-activator docking site is made up of residues from helices 3, 5, and 12, which form a hydrophobic channel conserved among the NR family members. The integrity of this surface is essential for co-activator binding and as a consequence the transactivation function (AF-2) of the LBD (reviewed in Ref. 2).

We have previously shown that a short alpha -helical sequence, the LXXLL motif, is necessary and sufficient for mediating the interaction of the co-activators RIP140, SRC1 and CBP/p300 with NRs (3). LXXLL motifs have also been shown to mediate binding of other co-activators including TIF1 (4), TIF2/GRIP1 (5, 6), p300/CBP-interacting protein/ACTR (7, 8), TRAP220/DRIP205 (9-11), PPARgamma co-activator-1 (12), and ASC-2/RAP250/NRC (13-15) to NRs. The number and sequence of LXXLL motifs varies considerably among the co-activators and is likely to account for observed differences in binding of co-activators to selected NRs or classes of NRs. The 160-kDa co-activators represented by SRC1, TIF2, and p/CIP each have a nuclear receptor interaction domain (NID) containing three LXXLL motifs. These sequences and the spacing between them are highly conserved, and we and others (3, 5, 7, 16, 17) have shown that they mediate high affinity binding to NRs. In addition, the SRC1a isoform has an additional LXXLL motif at its C terminus (16). The 140-kDa protein RIP140 contains nine functional LXXLL motifs that show variable affinity for the LBD of ERalpha (3). We have also shown that CBP (and its paralog p300) contains two conserved LXXLL motifs near the N terminus of the protein, which account for its weak interaction with ER and RAR (3). Some nuclear receptor-binding proteins such as NR-binding SET domain-containing protein contain variant motifs such as FXXLL (18). In addition, a leucine-rich helix (LXX(I/H)IXXX(I/L)) termed the co-repressor of nuclear receptor box mediates the binding of the silencing mediator of the co-repressors for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (NCOR) proteins to the unliganded forms of NRs (19-21).

The crystal structure of the LBD of PPARgamma in complex with a portion of the NID of SRC1 revealed that LXXLL motifs adopt an alpha -helical conformation that binds the AF-2 surface of the liganded LBD (22). A similar arrangement was observed in the crystal structures of the liganded TRbeta and ERalpha LBDs complexed with a peptide corresponding to motif 2 of GRIP1 (6, 23). The three conserved leucines were found to align on the face of the alpha -helix which packs against the hydrophobic channel on the LBD surface. Two charged residues that are highly conserved among LBDs (a glutamic acid in helix 12 and a lysine residue in helix 3) and that are especially critical for AF-2 function appear to act as a charge clamp to hold the LXXLL alpha -helix in position (22). In all three structures, both AF-2 surfaces in the LBD homodimer were occupied with one LXXLL alpha -helix. The PPARgamma /SRC1 NID structure indicates that, as a consequence of having multiple LXXLL motifs, a single p160 co-activator protein can contact both AF-2 surfaces in NR dimers. Consistent with this, we and others (5, 16, 24, 25) have found a requirement for at least two functional motifs to support high affinity binding of SRC1 and TIF2/GRIP1 NIDs or full-length proteins to steroid receptors. Similarly, the co-activator TRAP220/DRIP205, which was isolated on the basis of interaction with class II receptors TR and VDR, contains two functional LXXLL motifs (NR-1 and NR-2), which have been shown to make selective contacts with VDR and RXR in the VDR/RXR heterodimer (10, 11). A strong interaction was identified between NR-2 and VDR, whereas a weaker interaction was observed between the NR-1 motif and RXR. In contrast, other putative co-activators appear to contain a single functional LXXLL motif, e.g. TIF1 (4) and ASC-2/RAP250/NRC (13-15). Thus, the number and sequence of LXXLL core motifs, and possibly flanking sequences, will influence the selectivity and affinity of co-activators for different NRs.

In this study we have defined the minimal or core LXXLL sequence capable of binding to NRs. By comparing the interactions of LXXLL core motifs derived from three different co-activators with a panel of NR LBDs, we have identified elements of the LXXLL core sequence that influence the affinity and selectivity of co-activators for NRs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Plasmid Constructions-- The two-hybrid vectors pBL1 and pASV3 (2) expressing the human ERalpha DNA binding domain (DBD) and the VP16 acidic activation domain (AAD), respectively, were gifts from R. Losson and P. Chambon. The plasmid pCMV-beta CBP was a gift from R. Goodman. The plasmid pBSKS-mCBP-HA was a gift from A. Bannister and T. Kouzarides. DBD-LXXLL motif fusion proteins were generated by ligation of phosphorylated, annealed oligonucleotide pairs into the pBL1 vector. Fragments of RIP140 and CBP were generated by PCR using elongase or Taq polymerase and cloned into pBL1 or a modified GST-2TK vector containing cloning sites compatible with the PCR constructs. The constructs AAD-RARalpha (aa 200-462) and AAD-RXRalpha (aa 230-467) were generated by cloning PCR fragments into a modified pASV3. All fusion constructs were fully sequenced. The constructs pSG5-hSRC1e (16) GST-ERalpha LBD (previously termed GST-AF-2), AAD-ERalpha (aa 282-595), AAD-ERalpha Mut (ML-543/4-AA) (3), AAD-PR (aa 633-933), and AAD-AR (aa 625-919) (27) have been described previously.

Two-hybrid Interaction Assays-- The yeast reporter strain used for all two-hybrid assays was W303-1B (HMLalpha MATalpha HMRalpha his3-11, 15 trp1-1 ade2-1 can1-100 leu2-3, 11 ura3) carrying the reporter plasmid pRLDelta 21-U3ERE, which contains a lacZ gene driven by a modified URA3 promoter containing three estrogen response elements (28). Transformants containing the desired plasmids were obtained by selection for the appropriate plasmid markers and grown to late log phase in 15 ml of selective medium (yeast nitrogen base containing 1% glucose and appropriate supplements) in the presence or absence of up to 1 µM of the appropriate ligand (all-trans-retinoic acid; 9-cis-retinoic acid; 17-beta -estradiol; R5020; mibolerone) or an equivalent volume of vehicle. The preparation of cell-free extracts by the glass bead method and the measurement of beta -galactosidase activity in the extracts were performed as described previously (28). Two-hybrid experiments were performed three or more times, and reporter activities are expressed as nmol/min/µg protein.

Immunodetection of Proteins-- The expression of DBD fusion proteins in yeast cell-free extracts was monitored by immunodetection using a monoclonal antibody recognizing the human ER (a gift from P. Chambon, Strasbourg). The antibody recognizes the "F" region of the LBD in the human ER and also the F region tag at the N termini of the DBD fusion proteins (26). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western blotting. A peroxidase-linked goat anti-mouse IgG was used to detect the primary antibody.

GST Pulldown and Peptide Inhibition Assays-- GST fusion proteins were expressed in Escherichia coli DH5alpha using isopropyl-beta -D-thiogalactopyranoside induction as described previously (3, 16). GST-Sepharose beads were loaded with GST alone or GST fusion proteins prepared from bacterial cell-free extracts. 35S-Labeled mouse ERalpha and SRC1e proteins were generated by coupled in vitro transcription/translation from the plasmids pSP6-MOR (a gift from Roger White) and pSG5 hSRC1e (16) and tested for interaction with GST proteins as described previously (29). Binding was carried out overnight at 4 °C with gentle mixing in NETN buffer (50 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0) containing protease inhibitors in the presence or absence of 1 µM estradiol (E2) as required in a final volume of 1 ml. Peptides were dissolved in sterile water at a concentration of 4 mg/ml and added to GST-binding reactions at a final concentration of 50 µM, immediately before the addition of ligand.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Definition of a Minimal Functional LXXLL Sequence-- We have previously shown that a 14-residue synthetic peptide corresponding to the C-terminal LXXLL motif (motif 4) of SRC1e efficiently competed the ligand-dependent binding of in vitro translated SRC1 proteins to the LBD of ERalpha in GST pulldown experiments (3). This ability to compete the ERalpha /SRC1 interaction was shown to be dependent on the integrity of the LXXLL motif, as a peptide in which two conserved leucines (residues +4 and +5) were replaced with two alanines failed to interfere with SRC1 binding to ERalpha . In this study, we have used the peptide competition assay to define the minimal LXXLL sequence required to bind the LBD of ERalpha . A series of peptides spanning the high affinity binding motif 4 sequence were tested for their ability to compete the interaction of SRC1e with GST-ERalpha LBD. As shown in Fig. 1, the ligand-dependent recruitment of SRC1e to GST-ERalpha LBD was strongly inhibited by the peptides SLLQQLLTE and SLLQQLLT, respectively. However, omission of the serine (LLQQLLTE) or threonine (SLLQQLL) residues at positions -2 and +6 (relative to the first conserved leucine residue, designated +1) resulted in a strong reduction in the ability of the resulting peptides to compete SRC1e binding to ERalpha LBD. As expected, peptides in which the LXXLL motif is disrupted or truncated showed no competitive properties in these experiments. Similar results were obtained using the SRC1a isoform (data not shown). These results are consistent with our previous observation that as little as 8 amino acids comprising the LXXLL motif is sufficient to bind to the LBD of ERalpha in yeast two-hybrid assays (3) and define the core NR-binding sequence as -2 to +6.



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Fig. 1.   Mapping the LXXLL core motif. Glutathione-Sepharose-bound GST-ERalpha LBD was incubated with 35S-labeled in vitro translated SRC1e protein in the presence or absence of 1 µM 17-beta -estradiol (E2) as indicated. Binding was carried out in the presence or absence of competitor peptides (final concentration 50 µM) as indicated. The sequences of the competitor peptides are shown. The conserved leucine residues are boxed, and the amino acids are numbered with respect to the first conserved leucine residue.

LXXLL Motifs in SRC1 Display Differential Affinities for the LBDs of Nuclear Receptors-- The human SRC1 protein is expressed in a variety of cell lines as two major isoforms (SRC1a and SRC1e) which differ at their C termini (16). Both isoforms contain the nuclear receptor interaction domain (NID) consisting of three LXXLL motifs (motifs 1-3) that are conserved in other p160 co-activators. The SRC1a isoform contains a 4th motif at the extreme C terminus of the protein that is responsible for the ability of this region of SRC1a to interact with nuclear receptors (Figs. 1 and 2 and Refs. 3, 16, and 30).



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Fig. 2.   LXXLL motifs derived from SRC1 display differential interactions with steroid and retinoid receptor LBDs. Yeast two-hybrid experiment showing interaction of LXXLL motifs from SRC1a with the LBDs of steroid and retinoid receptors in yeast two-hybrid experiments. Control (DBD) is the pBL1 vector containing the human estrogen receptor DNA binding domain. Motifs 1-4 are bait constructs in which the DBD is fused in frame with amino acids 632-642, 689-699, 748-758, and 1434-1441 of SRC1a, respectively. The prey vectors consist of the acidic activation domain of VP16 (AAD) fused in frame to the ligand binding domains (LBD) of estrogen receptor alpha  (AAD-ERalpha ), the androgen receptor (AAD-AR), retinoic acid receptor alpha  (AAD-RARalpha ) and the retinoid X receptor (AAD-RXRalpha ). Plasmids encoding bait and prey proteins were co-transformed into the yeast strain L40 and cultured overnight in the presence or absence of 500 nM of cognate ligand, E2, Mibolerone, all-trans-retinoic acid (AT-RA), or 9-cis retinoic acid (9C-RA). Reporter activity in cell-free extracts is expressed as units of beta -galactosidase activity. Shaded columns indicate the reporter activity in the absence of ligand. Black columns indicate the reporter activity in the presence of cognate ligand.

Our previous results showed that upon addition of ligand, motifs 1-4 of SRC1 bind strongly to the LBD of ERalpha with similar apparent affinities, as determined by yeast two-hybrid experiments (3). In this study, we compared the interactions of SRC1 LXXLL motifs with the LBDs of hERalpha , hRARalpha , mRXRalpha , hAR, and hPR receptors. As shown in Fig. 2, motif 1 interacted strongly with the LBD of ERalpha as expected but displayed a significantly reduced ability to bind to the LBDs of RARalpha and AR. In addition, the reporter activation due to interaction of motif 1 with the LBDs of RXRalpha (Fig. 2) and PR (Fig. 5) was consistently reduced in comparison to the other motifs. The motif 2 sequence displayed significant ligand-dependent interaction with all LBDs tested, although a lower affinity for the AR LBD was observed in comparison to other NR LBDs tested (Fig. 2). Motifs 3 and 4 exhibited strong interaction with the panel of LBDs tested in the presence of ligand. However, motif 3 and to a lesser extent motif 4 displayed significant ligand-independent interaction with the LBD of RXRalpha . The significance of the ligand-independent interactions with RXRalpha LBD is unclear, as this was only observed in yeast two-hybrid experiments. No significant ligand-independent interaction was observed between GAL-RXRalpha and full-length SRC1, the intact NID or C terminus of SRC1a in transiently transfected mammalian cells (51).

High and Low Affinity LXXLL Motifs in RIP140-- We compared the interaction of RIP140 motifs L1 to L9 with the LBDs of ERalpha , RARalpha , and RXRalpha . As shown in Fig. 3, the L1-L9 core motifs displayed differences in the strength of reporter activation due to binding to ERalpha LBD. In general, similar patterns of interaction were observed for each core motif with the LBDs of ERalpha , RARalpha , and RXRalpha , with some exceptions as discussed below. By comparison of the reporter activities induced in the presence of ligand, we sought to determine how the sequences of individual core motifs influenced the apparent strength of interactions with NR LBDs in the two-hybrid system. Motifs showing strong NR binding in this assay generally contained a hydrophobic residue at position -1, usually leucine, valine, or tyrosine. However, the presence of a hydrophobic residue at position +2 (as in motifs L3 and L5) appeared to reduce affinity for NR LBDs. The motif L3 displayed weak interaction with the LBDs of ERalpha and RXRalpha but negligible binding to the RARalpha LBD. The motif L5, on the other hand, displayed weak interaction with the LBDs of ERalpha and RARalpha but no detectable binding to RXRalpha . Similar selectivity was observed using larger fragments of RIP140 spanning the L3 or L5 motifs, in yeast two-hybrid experiments (data not shown).



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Fig. 3.   Differential interaction of LXXLL motifs derived from RIP140 with the LBDs of ERalpha , RARalpha , and RXRalpha . Yeast two-hybrid experiments were performed as in Fig. 2. DBD-L1 through DBD-L9 represent bait constructs in which the DBD is fused in frame to RIP140 sequences, as indicated in parentheses. Shaded and black columns represent reporter activity in the absence and presence of 500 nM cognate ligand, respectively.

Low Affinity LXXLL Motifs Mediate the Interaction of CBP with NRs-- Several groups have observed ligand-dependent binding of N-terminal fragments of CBP to NRs (31-34). We compared the ability of in vitro translated SRC1 or CBP full-length proteins to bind to the LBD of ERalpha in GST pulldown experiments. SRC1e exhibited strong binding to the LBD of ERalpha in the presence of 17-beta -estradiol (Fig. 1). In contrast, full-length CBP showed only a very weak ability to bind the ER LBD in similar experiments (51).

Weak interaction of the N terminus of CBP with RARalpha has been localized to amino acids 1-101 (31, 32). In addition, a second region comprising amino acids 356-495 has been reported to bind weakly to RXRalpha , although this interaction was apparently independent of ligand (32). Analysis of the sequence of CBP revealed it to contain three potential LXXLL motifs (amino acids 68-78, 355-365, and 2067-2077), which are conserved in p300. Indeed, we have previously reported that the LXXLL motif located between amino acids 68 and 75 displays weak ligand-dependent binding to the LBD of ERalpha in two-hybrid assays. However, the strength of binding of this motif to ERalpha (as indicated by reporter activation) was between 10- and 50-fold lower than that achieved by motifs derived from SRC1 or RIP140, despite being expressed at comparable levels (3).

To examine interactions of CBP with NRs in more detail, PCR products spanning the regions 1-101, 345-381, and 1981-2165 (shown schematically in Fig. 4A) were fused in frame with a heterologous DBD. These constructs were used in yeast two-hybrid experiments to test for interactions with the LBDs of ERalpha , RARalpha , or RXRalpha (Fig. 4, B and D). The CBP N-terminal 1-101 construct displayed potent transcriptional activity in yeast, consistent with reports that the N terminus of CBP functions as a transactivation domain in mammalian cells (35-37). Reporter activation was slightly enhanced (~1.5-1.8-fold) upon addition of ligand (Fig. 4B). To determine whether the LXXLL motif mediates this weak ligand-induced interaction with NRs, the conserved leucine at residue 72 was mutated to alanine. This resulted in a loss of the weak ligand-induced response (Fig. 4B), supporting the hypothesis that this sequence is required for ligand-induced NR binding. Western blots confirmed that the wild type and mutant fusion proteins were expressed at similar levels (data not shown). The data shown in Fig. 4B were pooled with data from replicated experiments to conduct an analysis of variance test using the SPSS statistics package. The increased reporter activity due to interaction of DBD CBP-(1-101) with VP16-NRs in the presence of ligand was found to be significant (F(1, 11) = 13.007, p < 0.01). In addition the inability of the DBD CBP-(1-101) (L72A) to increase reporter activity due to interaction with VP16-NRs in response to ligand was also found to be significant (F(1, 11) = 37.309, p < 0.001).



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Fig. 4.   N- and C-terminal fragments of CBP interact weakly with the LBDs of ERalpha , RARalpha , and RXRalpha . A, schematic representation of CBP protein showing approximate locations of sequences encoding LXMs 1-3, the CH1, CH2, and CH3 domains, the bromodomain, and the histone acetyltransferase (HAT) domain. Known binding sites for transcription factors and other proteins are also shown. The black boxes represent fragments of CBP used in yeast two-hybrid experiments, below. B and D, yeast two-hybrid interaction of DBD-CBP-(1-101), DBD-CBP-(1-101) (L72A), DBD-CBP-(342-381), and DBD-CBP-(1891-2165) with AAD or AAD-LBDs, as indicated. Shaded and black columns represent reporter activity in the absence and presence of 500 nM cognate ligand, respectively. C and E, GST pulldown experiment showing interaction of 35S-labeled full-length ERalpha with GST, GST-CBP-(1-101), GST-CBP-(1-101) (L72A), GST-CBP-(342-381), and GST-CBP-(2058-2130) in the presence or absence of 1 µM E2, as indicated.

To confirm this weak interaction in vitro, the CBP-(1-101) sequence was fused in frame with GST and expressed in E. coli for use in pulldown experiments. As shown in Fig. 4C, CBP-(1-101) bound to full-length ERalpha in a ligand-dependent manner. In contrast, the CBP-(1-101) (L72A) mutant failed to bind to ERalpha LBD to a level above the controls. However, it should be noted that the efficiency of binding in the GST pulldown experiments was significantly lower than that achieved with GST-NID of SRC1 (data not shown), indicating that ERalpha binds SRC1 with a higher affinity than to CBP.

The second N-terminal fragment CBP-(345-381) also displayed weak ligand-inducible interaction with the LBDs of ERalpha , RARalpha , and RXRalpha in yeast two-hybrid experiments (Fig. 4D). In contrast to CBP-(1-101), no intrinsic reporter activation was observed with this construct. The C-terminal fragment CBP-(1891-2165) displayed a weak interaction with the LBDs of ERalpha and RXRalpha but not RARalpha in these experiments, enhancing the activity of the reporter 2-3-fold in the presence of ligand (Fig. 4D). GST-pulldown experiments confirmed the weak ligand-dependent interaction of CBP sequences 345-381 and 1891-2165 with full-length ERalpha (Fig. 4E). Note that the amount of ERalpha protein retained on the beads was less than 10% of the input.

We next determined whether the CBP LXXLL core motifs (designated CBP LXM-1-3) could bind the LBDs of steroid and retinoid receptors. We had previously demonstrated that CBP LXM-1 interacted weakly with the LBD of ERalpha . As shown in Fig. 5A, CBP LXM-1 and CBP LXM-3 displayed ligand-induced binding to RARalpha and PR. However, no significant interaction with the LBD of the androgen receptor (AR) was detected (Fig. 5A). Despite several attempts, we were unable to achieve stable expression of the construct DBD-CBP-(356-366) as determined by Western blots (data not shown). Thus, we were unable to determine whether LXM-2 is sufficient to mediate binding to NRs in the two-hybrid assay. However, the interaction of CBP-(345-381) fragment with the LBDs of ERalpha , RARalpha , and RXRalpha in this system (Fig. 4D) and in vitro (Fig. 4E) suggests that LXM-2 also functions as a weak NR-binding motif.



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Fig. 5.   LXXLL motifs mediate weak interaction of CBP with the LBDs of steroid and retinoid receptors. A, yeast two-hybrid interaction of CBP LXXLL motifs with AAD-RARalpha , AAD-ERalpha , AAD-RXRalpha , and AAD-AR, performed as in Fig. 2. LXM-1 and LXM-3 are constructs in which the DBD is fused in frame with amino acids 68-75 and 2067-2074 of CBP, respectively. Shaded columns indicate the reporter activity in the absence of ligand. Black columns indicate the reporter activity in the presence of cognate ligand. B, interaction of LXXLL motifs from SRC1 and CBP with the LBD of the progesterone receptor (AAD-PR), in the presence or absence of R5020 (500 nM) as indicated. C, Western blot showing expression of the DBD-LXXLL constructs in B. The antibody was a mouse monoclonal antibody derived against the F-region epitope of the human ERalpha which is present at the N terminus of the fusion proteins.

The fold activation of reporter activities achieved with the LXM-1 and LXM-3 core motifs (Fig. 5A) was slightly higher than those obtained due to interaction of LBDs with DBD-CBP-(1-101) (Fig. 4B) and DBD-CBP-(1891-2165) (Fig. 4D). As it has been shown that flanking sequences can influence the affinity of LXXLL motifs for NRs (47, 50), this may indicate that core sequences from CBP have a slightly increased affinity for steroid and retinoid receptors compared with larger CBP fragments. Nonetheless, as shown in Fig. 5B, the reporter activity due to interaction of LXXLL motifs derived from SRC1 with the LBD of the PR was 50-100-fold higher than that achieved with CBP motifs. This was not due to lower expression of the CBP fusion proteins, as shown by Western blots (Fig. 4C). Taken together, our results indicate that LXXLL motifs at the N and C termini of CBP and p300 account for the low affinity binding of these proteins to steroid and retinoid receptors.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we have defined the core LXXLL motif as eight amino acids, requiring two residues N-terminal to the first conserved leucine (+1) and at least one residue C-terminal to the 3rd conserved leucine (+5) (Fig. 6). Consistent with this, it has been previously observed that peptides of 13 and 14 amino acids in length encompassing motif 2 of GRIP1 strongly compete the interaction of the GRIP1 NID with the TRbeta LBD, whereas the hexapeptide LLRYLL did not (6). The crystal structure of the LBD of TRbeta in complex with a synthetic peptide corresponding to motif 2 of GRIP1 showed that the peptide adopts an amphipathic alpha -helix conformation of nearly 3 turns from Lys-688 to Gln-695. Residues outside this sequence were unstructured or in an extended coil conformation. In contrast, the peptide in solution was found to be in a random coil formation, thus alpha -helicity appears to be induced by the interaction with the TRbeta LBD. The alpha -helical structure in motif 2 induced by binding the TRbeta LBD involves amino acids KILHRLLQ and correlates exactly with the boundaries -2 to +6 that we have defined as the minimum length of a LXXLL motif sufficient for binding to AF-2. In addition, in a phage display screen for peptides that bind to liganded ERalpha LBD, more than 30% of the isolated LXXLL sequences commenced at position -2. The shortest C-terminal boundary isolated in the same screen terminated at +7 (38). Thus, we conclude that the minimal NR binding module is an 8-amino acid alpha -helix.



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Fig. 6.   Sequences of LXXLL motifs showing differential affinities for steroid and retinoid receptors. The sequences of the LXXLL motifs derived from SRC1, RIP140, or CBP, and their respective amino acid numbers are shown. The sequences are aligned with respect to the first conserved leucine residue (numbered 1). The conserved leucine residues and the amino acid at the -1 position are boxed.

A number of studies have indicated that co-activators show differential but overlapping binding preferences for NRs. Although there are reports of interactions of fragments of CBP with steroid and retinoid receptors, we and others (51) have found these interactions to be far weaker than the interaction of p160 proteins with steroid or retinoid receptors (Fig. 1 and Fig. 5D). Fluorescence resonance energy transfer experiments failed to show association of ERalpha and CBP in vivo, under conditions where the ERalpha /SRC1 and PPARgamma /CBP association was readily observed (39). Similarly, others (17, 40-42) have reported only very weak interactions between steroid, thyroid, and retinoid receptors with CBP. In contrast interactions of CBP and PPARs seem to be more robust, although still considerably weaker than PPARgamma /p160 interactions (39, 43-45).

We have demonstrated that LXXLL core motifs derived from SRC1a, RIP140, and CBP show distinct preferences for steroid and retinoid receptors. By analysis of the core motif sequences, it is apparent that positions -1 and +2 (and perhaps +3) have a strong impact on the ability of core motifs to bind to different NR LBDs. We noted that the LXXLL motifs showing the strongest interactions with the panel of LBDs selected tended to have a hydrophobic residue at position -1 (Fig. 6). For example, motifs 2 and 3 of SRC1/TIF2/ACTR, and motif 4 of SRC1a, all of which display high affinity for ERalpha and other NRs, contain a leucine or isoleucine residue at the -1 position. In addition, in an affinity selection of randomized peptides that bind the LBD of ERalpha , ~30% of LXXLL sequences selected contained a hydrophobic residue at -1 (38). In another study using a "focused" peptide library, 12/17 sequences affinity selected with ERalpha LBD contained a leucine or isoleucine residue at the -1 position (46). The SRC1 motif 1 has a lysine residue at position -1. Although this does not appear to affect its interaction with ERalpha in our experiments, motif 1 displayed significantly reduced binding to RARalpha , RXRalpha , AR, and PR (Fig. 2). Consistent with this, it has been shown that a peptide based on motif 1 sequence was inferior to other SRC1 LXXLL motif peptides in competition of ERalpha /SRC1 interaction in vitro (47).

CBP has been reported to interact weakly with RAR and RXR via the N-terminal 400 amino acids (Fig. 5; see Refs. 31 and 32). However, it has been shown that deletion of the first 400 residues of CBP did not affect its ability to bind RAR (31, 41). This suggested that additional binding site(s) distal to the N terminus are present in CBP or that CBP interaction with NRs can be indirect. CBP contains three LXXLL motifs, and we have demonstrated here that these mediate weak ligand-dependent interactions of the N- and C-terminal fragments of CBP with steroid and retinoid receptors. None of the LXXLL sequences in CBP contain a hydrophobic residue at position -1. In a recent study of interaction of these sequences with the LBDs of PPARgamma and RXRalpha , the CBP LXM-1 motif was shown to bind to PPARgamma but not RXRalpha (45). Furthermore, the same authors reported that the replacement of the glutamine at -1 with leucine significantly increased the ability of CBP LXM-1 to bind RXRalpha . This is consistent with our finding that co-activators with high affinity for steroid and retinoid receptors generally contain LXXLL motifs with a hydrophobic residue at position -1. Thus, while we and others (40, 48, 51) have shown CBP is required for the function of steroid and retinoid receptors, we propose that at least some NRs recruit CBP indirectly via the p160s.

Although 8 of the 9 nine LXXLL motifs present RIP140 have a hydrophobic residue at position -1 (the exception being L4), not all demonstrate high affinity binding to the panel of LBDs tested. For example, we noted that motif L5 of RIP140 has very low affinity for NRs, despite satisfying the requirement in having a leucine at position -1 (Fig. 3). This may be due to the presence of a leucine residue at position +2, which is rare among the LXXLL motifs identified to date. The presence of a bulky hydrophobic side chain at this position may be detrimental to the formation of an amphipathic alpha -helix. However, there are examples of core motifs that tolerate a hydrophobic residue at +2, notably motif 1 of SRC1 which shows reduced affinity for NRs, and the functional LXXLL motif of human ASC-2/NRC/RAP250, which has a valine at position +2 (13-15). Further studies will be required to determine the repertoire of amino acids tolerated within core motif sequences.

Structural studies have shown that lysine and glutamic acid residues in the LBD that are almost universally conserved among the NR family may act to clamp to the LXXLL alpha -helix at the AF-2 surface (6, 22, 23). This involves contacts with residues outside the core motif. In addition, it has been shown that CBP can acetylate a lysine residue just upstream of motif 1 in ACTR and that this modification appears to reduce the binding of ACTR to NRs (49). In addition, reports by several groups (47, 50) indicate that sequences flanking the core motif affect the affinity and selectivity of co-activators for NRs. Thus, sequence flanking the core motif may also influence the interaction of co-activators with NRs. Understanding the determinants of LXXLL motif/LBD interactions will facilitate the design of peptides and other antagonists capable of disrupting specific NR/co-activator interactions.


    ACKNOWLEDGEMENTS

We are grateful to Regine Losson, Pierre Chambon, Richard Goodman, and Tony Kouzarides for gifts of plasmids and antibodies and Graham Clark (ICRF) for automated sequencing. We also thank Eric Kalkhoven, Charlotte Bevan, and Frank Claessens for useful discussions and Paula Moran for advice on statistical analysis.


    FOOTNOTES

* This work was supported by the Wellcome Trust, the Imperial Cancer Research Fund, and a Marie Curie fellowship (to D. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-116-252-3474; Fax: 44-116-252-3369; E-mail dh37@le.ac.uk.

Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M009404200


    ABBREVIATIONS

The abbreviations used are: LBD, ligand binding domain; SRC1, steroid receptor co-activator; RIP140, receptor interacting protein; CBP, CREB-binding protein; NR, nuclear receptor; AF, activation function; TIF, transcriptional intermediary factor; GRIP1, glucocorticoid receptor interacting protein; ACTR, nuclear receptor co-activator; TRAP220, thyroid receptor-associated protein 220 kDa; DRIP205, vitamin D receptor interacting protein 205 kDa; ASC-2, activating signal co-integrator; RAP250, nuclear receptor activating protein 250 kDa; NRC, nuclear receptor co-activator; NID, nuclear receptor interaction domain; LXM, LXXLL motif; ER, estrogen receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor, AR, androgen receptor; PR, progesterone receptor; PPAR, peroxisome proliferator activated receptor; VDR, vitamin D receptor; AAD, acidic activation domain; DBD, DNA binding domain; PCR, polymerase chain reaction; GST, glutathione S-transferase; E2, 17-beta -estradiol; aa, amino acids.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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