Molecular Basis of Agonistic Activity of ERα17p, a Synthetic Peptide Corresponding to a Sequence Located at the N-Terminal Part of the Estrogen Receptor αLigand Binding Domain

Abstract

ERα17p is a synthetic peptide corresponding to a regulatory motif located within the autonomous AF-2a region of the estrogen receptor α (ERα). ERα17p binds to the receptor and enhances its transcriptional activity. Structural characteristics of this peptide suggest that it interferes with intra- and/or interprotein interactions involving ERα.

Full text

346 Letters in Drug Design & Discovery, 2007, 4, 346-355
Molecular Basis of Agonistic Activity of ER 17p, a Synthetic Peptide
Corresponding to a Sequence Located at the N-Terminal Part of the
Estrogen Receptor Ligand Binding Domain
Dominique Gallo1, Yves Jacquot2, Anny Cleeren1, Françoise Jacquemotte3, Ioanna Laïos1, Guy
Laurent4 and Guy Leclercq*,1
1Laboratoire J.-C. Heuson de Cancérologie Mammaire, Université Libre de Bruxelles (U.L.B.), Institut Jules
Bordet, 1 rue Héger-Bordet, B-1000 Brussels, Belgium
2Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7613, "Synthèse, Structure et Fonction de Molécules
Bioactives"; FR 2769, Case courrier 45, 4 place Jussieu, F-75005 Paris, France
3Département des Substances Naturelles et de Biochimie, Institut Meurice, 1 avenue Emile Gryzon, B-1070
Brussels, Belgium
4Service d’Histologie et de Cytologie Expérimentale, Faculté de Médecine et de Pharmacie, Université de Mons-
Hainaut, 6 avenue du Champ de Mars, B-7000 Mons, Belgium
Received February 07, 2007: Revised March 17, 2007: Accepted March 24, 2007
Abstract: ERα17p is a synthetic peptide corresponding to a regulatory motif located within the autonomous
AF-2a region of the estrogen receptor α (ERα). ERα17p binds to the receptor and enhances its transcriptional
activity. Structural characteristics of this peptide suggest that it interferes with intra- and/or interprotein
interactions involving ERα.
Keywords: Estrogen Receptor, Synthetic Peptide, ERα17p, Regulatory Motif, AF-2.
INTRODUCTION and T311 [6, 7]) and acetylation (K302 and K303 [8, 9]).
Motifs for SUMOylation [10], calmodulin (CaM)
association [11, 12, 13], proteolysis [14] and nuclear
localization [15, 16] have also been identified in this PPII
rich subregion.
Small synthetic peptides harboring a canonical motif
have proven to be valuable tools for investigating both
functions and conformational changes of proteins [1, 2] as
well as for developing new therapeutic approaches. In the
context of the treatment of estrogen receptor alpha (ERα)-
associated diseases such as breast cancer, one may anticipate
that ERα-derived peptides may open new avenues,
especially when pharmacological ligands appear devoid of
efficacy (i.e. antiestrogen resistance). In a similar way,
constitutive activity of orphan estrogen-related receptors
(ERRs), which is known to underlie a number of
pathological disorders (for review, see [3]), may perhaps be
abrogated or made sensitive to physiological regulation.
We recently reported that a synthetic peptide (i.e.
P295LMIKRSKKNSLALSLT311) encompassing the first
PPII rich subregion and containing the CaM binding site
elicits pseudo estrogenic responses in cell culture [13].
Indeed, this peptide-called ERα17p - induced the expression
of an estrogen-regulated reporter gene, caused ERα down
regulation and stimulated the growth of ERα-positive breast
cancer cell lines. The fact that ERα17p was devoid of
activity on ERα-negative cells clearly indicated that it acts
via a pathway involving ERα. The additional finding that
an ERα mutant lacking the P295-T311 motif exhibits a
constitutive transcriptional activity reveals that this motif is
implicated in inhibitory molecular interaction(s). Hence, the
agonist-like activity of ERα17p could stem from the
abrogation of such repressive mechanism(s).
The N-terminal part of the ligand binding domain (LBD;
E domain) of ERα, now identified as an autonomous
activation function (AF-2a), is a platform playing an
important role in receptor-mediated transcription [4].
According to our structural analyses, the S301-E330 sequence
of this domain harbors two polyproline II (PPII) rich
subregions (i.e. S301-T311 and L320-E330) that may interact
with a PPII recognizing domain (PRD) spatially located
close to the AF-2 coactivator binding site (end of helix H3,
βII-turn, helices H4, H5, H6 and the S1-S2 β-hairpin) [5].
These potential intramolecular interactions could contribute
to the regulation of ERα since the S301-T311 sequence
contains key aminoacids subjected to phosphorylation (S305
Data reported in the present paper support this view and,
moreover, unravel a previously unknown molecular
mechanism of ERα regulation. The current study thus
provides new guidelines for the design of drugs targeting
ERα-mediated processes.
MATERIAL AND METHODS
Chemicals
*Address correspondence to this author at the Laboratoire J.-C. Heuson de
Cancérologie Mammaire, Institut Jules Bordet, 1 rue Héger-Bordet, B-
1000 Brussels, Belgium; Tel: +3225413744; Fax: +3225413498; E-mail:
lcanmamm@ulb.ac.be
[2,4,6,7-3H] 17β-estradiol ([3H]E2; ~100 Ci/mmol) was
purchased from GE Healthcare (Little Chalfont,
Buckinghamshire, UK). MG-132 was obtained from
1570-1808/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

Properties of an ER -Derived Synthetic Peptide Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 347
Calbiochem (San Diego, CA). Estradiol (E2) and 4-
hydroxytamoxifen (OH-tam) were from Sigma (St Louis,
MO) and fulvestrant (ICI 182,780) was from Tocris
(Ellisville, MO). Tamoxifen was from Active Motif
(Carlsbad, CA). Steroid cofactor peptide derived from SRC-
1 (reference 60199-1) was obtained from Anaspec (San Jose,
CA).
Measurement of [3H]E2 Binding Capacity
Competition Binding Assays
Purified hERα adsorbed onto hydroxylapatite (Bio-Rad;
Hercules, CA), in 10 mM Tris-HCL buffer pH 7.5
containing 100 mM KCl and 1mg/ml BSA, was incubated
with 5 nM [3H]E2 in the absence or presence of increasing
concentrations of E2 or ERα17p (4 °C, overnight). Non-
specific binding was established by a parallel incubation
with a 200-fold excess of unlabeled E2. After washing of
hydroxylapatite pellets, bound [3H]E2 was extracted with
ethanol and measured using a Wallac 1409 liquid
scintillation counter (Perkin Elmer; Boston, MA). Specific
binding was calculated from the difference between
radioactivity levels measured in the absence and presence of
excess unlabeled E2.
Peptide Synthesis
ERα17p (H2N-PLMIKRSKKNSLALSLT-COOH) was
produced in the Natural Substances Laboratory of Meurice
Institute according to the Atherton and Sheppard solid phase
peptide synthesis method [17] on an Advanced ChemTech
90 apparatus (for experimental details, see [13]).
Biotinylated ERα17p was produced in order to link the
peptide to streptavidin-agarose beads (UltraLink
Immobilized Streptavidin Plus from Pierce, Rockford, IL).
Peptide synthesis was performed according to the same
procedure. Before cleavage, biotin-COOH (Novabiochem,
San Diego, CA) was coupled to the N-terminal proline with
HBTU.
Scatchard Plot Analysis
Hydroxylapatite-adsorbed purified hERα was incubated
with increasing concentrations of [3H]E2 (from 0.5 to 10
nM) in the absence or presence of 10 µM of ERα17p (4 °C,
overnight); non-specific binding was established by a
parallel incubation with a 200-fold excess of unlabeled E2.
[3H]E2 extraction and radioactivity measurement were carried
out as described above. [3H]E2 binding parameters (Bmax,
number of binding sites; Kd, dissociation constant) were
determined by Scatchard plot analysis [18].
Far-UV CD Spectroscopy
Far-UV CD spectra were recorded on a Jobin Yvon CD6
spectropolarimeter. ERα17p samples were prepared by
dissolving the peptide in 1 mM potassium phosphate buffer
adjusted to pH 7.0 (final peptide concentration ≈ 200 µM).
2,2,2-trifluoroethanol (TFE) 50 % or guanidine
hydrochloride 7.0 M were added to samples to enrich the
proportion of α-helix or PPII, respectively. Samples, in a
250 µl cuvette, were allowed to equilibrate for 5 minutes at
5 °C in the spectropolarimeter prior to data recording.
Measurements were carried out at 1 nm intervals over a
wavelength range from 185 to 260 nm. Reported spectra
correspond to averages of 4 scans with smoothing and
corrected for solvent/buffer contributions. Data were treated
with Dichrograph Software vers. 1.2. Mean residue
ellipticity [ ] was expressed in deg.cm2.dmol-1 from the
equation [ ] ≈ 3298∆ε, where ∆ε corresponds to the molar
ellipticity.
Assessment of ER -LxxLL Motif Association
Association of purified hERα with LxxLL-containing
peptide was assessed by ERα Elisa NR peptide (Active
Motif). Briefly, purified hERα (10 ng in diluent buffer) was
incubated with E2, tamoxifen, fulvestrant or ERα17p (all
compounds at 25 µM, 30 minutes on ice; according to
manufacturer’s instructions) before addition to a 96-well
plate coated with an LxxLL-containing peptide; controls were
run in parallel in the absence of any compound. Binding of
ERα to the plate was measured by colorimetry (dual
wavelength measurement at 450 nm with background
wavelength correction set at 600 nm), after addition of an
anti-ERα primary and of an HRP-conjugated secondary
antibody, according to the manufacturer's instructions.
ER 17p-Induced Responses in MCF-7 Cells
Interaction of ER 17p with Purified ER in Cell-Free
Conditions Cell Culture
MCF-7 and MVLN cells were propagated at 37 °C (5 %
CO2, humid atmosphere) in Earle’s based minimal essential
medium (EMEM) supplemented with Phenol Red, 2 mM L-
glutamine, 100 U/ml penicillin,100 µg/ml streptomycin and
10 % heat-inactivated fetal bovine serum (FBS) (all reagents
from Invitrogen/Gibco). All experiments were performed in
Phenol Red-free EMEM containing 10 % charcoal-stripped
FBS (change of medium 48 hours before assays).
ER Pull-Down Experiments
Purified human recombinant ERα (hERα; Calbiochem)
(~2.5 pmol in 1 ml of 50 mM Tris-HCl pH 7.5 buffer,
containing 150 mM NaCl, 0.5 % NP-40, 50 mM NaF, 0.1
mM orthovanadate, 0.6 mM PMSF, 0.3 mM TPCK and 1
mg/ml BSA) was incubated for 2 hours at 4 °C with either
ERα17p-linked beads or free agarose-streptavidine beads
(non-specific binding) (100 µl, 50 % slurry); ERα17p at 10
µM was added to the preparation as an additional control of
binding specificity. After washing, beads were suspended in
50 µl electrophoresis buffer (LDS Sample 1X buffer from
Invitrogen; Carlsbad, CA) and boiled for 5 minutes. ERα
levels were then measured by Western blot analysis (see [13]
for details) using F-10 anti-ERα antibody (Santa Cruz
Biotechnology; Santa Cruz, CA); control input = 1.5 %.
[3H]E2 Binding Whole Cell Assays
Binding Parameters
MCF-7 cells plated in 24-well dishes were incubated for
15 minutes with 10 µM ERα17p. After removal of the
medium, cells were exposed to increasing concentrations of
[3H]E2 (from 0.05 to 1 nM) for 45 minutes without or with
a 500-fold excess of unlabeled E2 (for assessment of non

348 Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 Gallo et al.
specific binding); two additional wells were used for
assaying protein concentration in cell extracts (see [13]).
Bmax and Kd parameters were established by Scatchard plot
analysis from the measurement of cell-associated [3H]E2.
fulvestrant at 0.1 µM. Cells were then washed 2 times with
PBS and luciferase activity was measured in cell lysates by
luminometry using Luciferase Assay System (Promega;
Madison, WI) according to a protocol described previously
[23].
Dissociation of [3H]E2-ERα Complexes
RESULTS
MCF-7 cells plated in 24-well dishes were incubated in
serum-free condition for 45 minutes with [3H]E2 at 1 nM
without or with a 500-fold excess of unlabeled E2 (for non-
specific binding) in the presence of 1 µM of MG-132.
Medium was then replaced for a further incubation (up to
240 minutes in fresh medium containing MG-132) in the
absence or presence of 1 nM E2 or 10 µM ERα17p. Rate of
[3H]E2-ERα dissociation was then determined by measuring
the time-course disappearance of cell-associated radioactivity.
Secondary Structure of ER 17p
Structural studies based on X-ray crystallographic data
revealed that the S301KKNSLALSLT311 sequence of ERα
harbors a partial PPII / α-helix mixed secondary structure
[5]. Could the biochemical activity of ERα17p, which
contains this sequence, be interpreted in terms of interactions
involving such a PPII structure? In order to answer this
question, far-UV CD spectrum of ERα17P was recorded in
1 mM phosphate buffer at 5 °C. These experimental
conditions were selected to minimize interconvertions
between peptide conformations on a short time scale. As
shown in Fig. (1), a strong negative band at 197 nm was
observed, reflecting a statistical-coil conformation. A weak
minimum at 219 nm was also detected suggesting the
occurrence of α-helix. In order to confirm this interpretation,
measurements were performed in 50 % TFE, since the latter
compound is known to enhance peptide helicity. [24, 25]. In
this experimental condition, two α-helix–related negative
bands at 200 and 222 nm ([ ]200 = -49271 deg. cm2. dmol-
1; [ ]222 = -63298 deg. cm2. dmol-1) were found.
Conversion of statistical-coil into α-helix was estimated
complete in the presence of TFE (from the equation: % α-
helix = -100([ ]222+3000)/33000 [26, 27]).
Assessment of Ser118 Phosphorylation in ER
MCF-7 cells were exposed to E2 at 1nM or ERα17p at 1
and 10 µM for 15, 30 and 240 minutes. Cells were then
lysed and the resulting extracts were submitted to Western
blot analysis (for experimental details, see [13]) using a
primary antibody recognizing ERα phosphorylated on
Ser118 (sc-12915 from Santa Cruz Biotechnology; Santa
Cruz, CA) (diluted 1 : 1000), followed by an anti-goat
secondary antibody (Pierce) (diluted 1 : 2000). Immunoblots
were visualized using a FLA-3000 camera (Fuji; Tokyo,
Japan).
Measurements of ER Transcriptional Activity
Progesterone Receptor mRNA Assessment by Nucleic Acid
Sequence Based Amplification (NASBA)
MCF-7 cells were exposed to 1 nM E2 or 10 µM
ERα17p for 6, 24 and 48 hours; control cells were
maintained in the absence of any compounds. Cells were
then harvested and their RNAs were extracted with TriPure
(Roche Applied Science; Mannheim, Germany) for NASBA
analysis [19, 20] (kindly performed by BioMérieux; Lyon,
France). Briefly, 5 ng of RNA were added to 10 µl of
NASBA buffer (final concentrations in 20 µl reaction
mixture: 40 mM Tris HCl, pH 8.5, 12 mM MgCl2, 70 mM
KCl, 5 mM dithiothreitol, 15 % v/v DMSO, 1 mM dNTP,
2 mM NTP, 0.2 µM of PgR and PPIB primers and 0.1 µM
of specific PgR and PPIB molecular beacons). Samples were
preincubated successively at 65 °C and 41 °C (2 minutes
each). Five µl of enzyme mix (0.08 U RNase H, 32 U T7-
RNA polymerase, 6.4 U RT) were then added for a further
incubation at 41 °C for 90 minutes. PgR mRNA levels were
normalized with respect to PPIB mRNA levels.
Since statistical-coil and PPII conformations share
similarities that extend to far-UV CD spectra, experiments
were reproduced in 7 M guanidine hydrochloride, a
chaotropic agent that forces peptides to adopt a helical PPII
extended structure. Addition of guanidine hydrochloride to
the buffer led to the emergence of a negative band at 212 nm
as well as a weak positive band at 220 nm (Fig. (1) insert).
Fig. (1). Far-UV CD spectra of ER 17p. Peptide was diluted (≈
200 µM) in 1 mM phosphate buffer pH 7. Spectra were recorded
at 5 °C without any addition (o), with 50 % TFE (∆) or 7 M
guanidine hydrochloride (insert).
pS2 mRNA Measurement by Northern Blot Analysis
MCF-7 cells were treated with E2 or ERα17p as
described above. RNA was extracted and submitted to
Northern blot analysis for pS2 mRNA and 28S rRNA levels
assessment (see [21]). Optical densities (ODs) were measured
using a GS-710 Calibrated Imaging Densitometer (Bio-Rad).
pS2 mRNA OD values were normalized relative to 28S
rRNA levels.
ERE-Dependent Transcription Measurement by Luciferase
Induction Assay
MVLN cells (MCF-7 stably transfected with pVit-tk-Luc
reporter plasmid [22]) were treated for 24 h with ERα17p at
10 µM and/or E2 at 0.1 nM, OH-tam at 0.1 µM or

Properties of an ER -Derived Synthetic Peptide Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 349
These two bands being undetectable in absence of the
chaotropic agent, it became evident that the strong negative
signal recorded at 197 nm in phosphate buffer could only be
due to a non-structured conformation.
ERα17p can associate with the receptor. This assumption
was demonstrated by pull-down experiments. Fig. (2A)
shows that, in a purified hERα preparation, the receptor
binds to ERα17p-linked beads (lane 4). Addition of
ERα17p in excess abrogates this binding (lane 3),
confirming the validity of our assay. Of note and for
unknown reason, ligands (i.e. E2, OH-tam and fulvestrant),
produced weak and spurious interferences with ERα17p-
ERα association (data not shown). On the other hand,
additional studies showed that ERα17p had only a weak
influence on E2-hERα association in this preparation of
purified receptor. Thus, conventional competitive curve (Fig.
(2B)), as well as Scatchard plot analysis (Fig. (2C)),
indicated that ERα17p at 10 µM barely affected the capacity
of purified hERα to bind [3H]E2 (11 % loss of Bmax
without any change of Kd value).
Hence, in media used for assessing its biological
properties, ERα17p adopts a statistical-coil conformation
with a weak α-helix contribution. However, a PPII structure
cannot be excluded since unfolded peptides are highly
dynamic and represent an energy-weighted ensemble of
conformations among which PPII may emerge [28, 29, 30,
31, 32]. Moreover, as described for other peptides [33, 34],
interaction of ERα17p with cognate protein domains may
strongly displace the equilibrium existing between its
various conformations in favor of a PPII structure.
ER 17p-ER Interaction Ligands that activate ERα enhance its association to
proteins or peptides harboring a canonical LxxLL motif [35,
36]. Ability of ERα17p to act similarly was analyzed by an
If the S301-T311 sequence in ERα is involved in a PPII /
PRD-based intramolecular interaction, then that implies that
Fig. (2). ER 17p - ER association. A. Pull down experiment: Purified hERα (input) was incubated with streptavidin-agarose beads
(non specific ; NS) or ERα17p-linked agarose beads in the absence (Control) or presence of free ERα17p (+ERα17p). Levels of bound
hERα as well as 1.5 % of total hERα (input) were assessed by Western blot. The figure refers to an immunoblot representative of four
independent experiments. B. [3H]E2 competition binding assays on hERα: Purified hERα adsorbed onto hydroxylapatite was
incubated with [3H]E2 in the absence (100 %) or presence of increasing amounts of E2 or ERα17p ; bound [3H]E2 was then measured.
The figure refers to an experiment performed three times. C. [3H]E2 binding parameters on ERα: Purified hERα adsorbed onto
hydroxylapatite was incubated with increasing concentrations of [3H]E2 (from 0.5 to 10
nM) in the absence (Control) or presence of
ERα17p at 10 µM. [3H]E2 binding parameters were then determined by Scatchard plot analysis. The figure refers to a representative
experiment performed three times. (control: Kd = 0.55 nM and Bmax = 83 fmol ; ERα17p: Kd = 0.58 nM and Bmax = 74 fmol).

350 Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 Gallo et al.
Fig. (3). Influence of ER 17p and ligands on ER binding to LxxLL-containing peptide. Purified hERα was incubated with (A) E2
and/or ERα17p or (B) E2 and/or tamoxifen (tam) or fulvestrant (fulv), all compounds at 25 µM. ERα-LxxLL complexation was then
assayed by the ERα Elisa NR peptide procedure (Active Motif). Data refer to the mean value (± SD) of three independent experiments.
Elisa-based procedure (ERα Elisa NR peptide from Active
Motif). As expected, preincubation of purified hER with E2
increased the binding of the receptor to the plate coated with
a LxxLL-containing peptide (Fig. (3A)). Surprisingly,
ERα17p decreased the binding of the hormone-free receptor
and, moreover, abrogated the enhancing effect of E2 on
receptor binding in this system. The potential induction of
an antagonistic conformation was rejected because tamoxifen
and fulvestrant failed to similarly reverse the effect of E2
(Fig. (3B)). The fact that a control LxxLL-containing peptide
derived from SRC-1 (LTERHKILHRLLQE), at same
concentration, acted like ERα17p (percent of control, E2 =
290 ± 24, peptide = 27 ± 18, E2 + peptide = 46 ± 5;
experiment performed three times) suggested that the
inhibitory effect of the latter resulted form a specific
interference with the LxxLL recognizing domain. Lys362 of
ERα implicated in LxxLL binding [37] is, indeed, located
close to residues potentially involved in the association
between the P295-T311 sequence and the β-turn/H4 region
(i.e. Pro365, Gly366 and Asp369 [5]).
E2- and ER 17p-Induced Molecular Events in MCF-7
Cells
Exposure of MCF-7 cells to ligands produces a gradual
decrease of the capacity of ERα to bind [3H]E2 [38, 39].
Such a loss was also recorded with ERα17p after a short-
time incubation (15 minutes; Fig. (4)). The fact that this
phenomenon occurs despite a proteasome blockade [13]
(coincubation with MG-132) excludes the possibility that it
Fig. (4). Effect of ER 17p on [3H]E2-ER complexation in MCF-7 cells. Cells were incubated during 15 minutes in the absence
(control) or presence of ERα17p at 10 µM. [3H]E2 binding parameters (Kd, Bmax) were then determined by Scatchard plot analysis.
The figure is representative of three experiments where Kd ranged from 0.07 to 0.22 nM.

Properties of an ER -Derived Synthetic Peptide Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 351
Fig. (5). Effect of ER 17p and E2 on [3H]E2-ER complex dissociation in MCF-7 cells. [3H]E2 labeled cells were maintained for up
to 240 minutes in the presence of MG-132 at 1 µM without or with E2 at 1 nM or ERα17p at 10 µM. At the end of each incubation
periods, residual [3H]E2 levels were measured. The figure refers to an experiment performed twice in triplicate.
might only be due to receptor degradation. Release of bound
chaperones (notably heat-shock proteins) as well as cofactor
recruitment are more likely explanations since, as shown
above, the ERα17p-induced loss of [3H]E2 binding capacity
is marginal in the case of purified hERα. Hence, in MCF-7
cells, ERα17p may trigger events associated with ERα
transactivation without directly interacting with the hormone
binding pocket. As shown hereunder, conformational
changes associated with these events seem also to affect the
stability of preformed [3H]E2-ERα complexes.
associated [3H]E2 indicates that the peptide may accelerate
the dissociation process, like what is seen with E2 (see Fig.
(5)) and other conventional ligands [42]. However, unlike
such ligands, ERα17p does not directly interact with the
hormone binding pocket, suggesting that it rather acts at a
step downstream of the occupation of this pocket. The
assessment of ERα phosphorylation supports this concept.
ERα phosphorylation at Ser118 subsequent to E2-
induced stimulation [43, 44] is an early step of a complex
mechanism [45, 46, 47, 48] leading in fine to enhanced
expression of estrogen-regulated genes (e.g. the progesterone
receptor; PgR, and the trefoil factor 1; TFF1 / pS2). As
expected, E2-induced events described here over were found
to be associated with a rapid phosphorylation of Ser118
(Fig. (6)). Remarkably, such a phosphorylation did not
occur in the case of ERα17p-induced stimulation,
suggesting that ERα17p operates at a later step of the
activation mechanism. As a consequence of this bypassed
process, ERα17p enhanced the transcription of PgR and pS2
despite the absence of estrogenic ligands (Fig. (7)).
In MCF-7 cells maintained in cold medium after
exposure to [3H]E2, cell-associated radioactivity showed an
abrupt decline during the first two hours after [3H]E2
withdrawal, followed by a flat curve (Fig. (5); experiment
performed in the presence of MG-132 to avoid any
proteasomal degradation of the receptor). The existence of
various [3H]E2-ERα complexes with distinct stabilities, due
to early LxxLL-induced changes of the hormone binding
pocket conformation [40], may explain this biphasic profile.
A potential dissociation of these complexes [41] with a
subsequent transfer of the labeled hormone to residual and/or
neosynthesized receptors, the binding capacity of which is
stabilized by chaperones, is another explanation. The finding
that the addition of ERα17p to the chase medium after
[3H]E2 withdrawal further decreased the level of cell-
Further studies conducted with MVLN cells indicated
that the transcription of an ERE-dependent reporter gene
enhanced by E2 was not amplified by ERα17p (Fig. (8)),
reinforcing the concept that the peptide acts via the same
Fig. (6). Influence of ER 17p on phosphorylation of Ser118 in ER . MCF-7 cells were cultured for 15, 30 and 120 minutes in the
absence (control ; a) or presence of E2 at 1 nM (b) or ERα17p at 1 (c) and 10 µM (d). Phosphorylation was assayed by Western blot
using anti-ERα Phospho Ser118 antibody (sc-12915). Immunoblot is representative of three independent experiments.

352 Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 Gallo et al.
Fig. (7). Effect of ER 17p on ER -dependent transcription. MCF-7 cells were cultured in absence or presence of E2 at 1 nM or
ERα17p at 10 µM for 6, 24 and 48 hours. A. PgR mRNA level was measured by NASBA. Mean values of two independent experiments
were normalized according to PPIB mRNA. B. pS2 mRNA level was evaluated by Northern blot. Columns represent mean OD of two
independent experiments. Data were normalized according to 28S rRNA.
pathway as the hormone. The finding that antiestrogens (i.e.
4-hydroxytamoxifen and fulvestrant) totally abrogate the
stimulatory effect of both compounds supports this view.
focuses on a peptide corresponding to a regulatory ERα
motif (ERα17p; P295-T311) provides valuable information
concerning ERα transactivation process.
DISCUSSION
There are at least two pools of ERα giving rise to either
a non-genomic or a genomic response (i.e. membrane-
associated and intracellular receptor pools, respectively) [49,
50, 51]. Although the translocation of ERα17p across the
cell membrane has not been experimentally analyzed, we
believe that this peptide mainly interferes with processes
mediated by intracellular receptors. Previous studies from
our laboratory [52] have indeed shown that the estrogenic
Pharmacological investigation on synthetic peptides
designed to reproduce protein motifs is not only an attractive
approach for the discovery of new drug candidates, but also
contributes to generate new investigative tools for
biomedical research. In this regard, our present study which
Fig. (8). Effect of E2, OH-tam and fulvestrant on the enhancement of ERE-dependent transcription by ER 17p. MVLN cells were
incubated for 24 hours in the absence (control) or presence of ERα17p at 10 µM, E2 at 0.1 nM, OH-tam at 0.1 µM or fulvestrant at 0.1
µM, alone or in combination. Luciferase activity was assayed in cellular extracts by luminometry and emitted light signals were
expressed in arbitrary units (relative luciferase units, RLU) per mg protein. Data refer to the mean value (± SD) of three independent
experiments with measurements performed in duplicate and are given as percentage of control.

Properties of an ER -Derived Synthetic Peptide Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 353
activation of membrane receptors neither decreases the E2
binding capacity of cells nor induces enhanced ERE-
dependent transcription like what is observed with ERα17p.
On the other hand and as stated here, Ser118
phosphorylation which usually results from both E2 and
growth factor-induced stimulations (see [53]), does not occur
after ERα17p treatment. This lack of phosphorylation
excludes the participation of the peptide in MAPK-involved
crosstalks initiated at the cell membrane.
that ERα17p operates without enhancing phosphorylation of
Ser118 which locates close to AF-1, supports this view.
The P295-T311 sequence of ERα is located within the
autonomous AF-2a region of the receptor [4]. Hence, partial
disruption induced by ERα17p of inhibitory intra- and
intermolecular interaction(s) to which this region contributes
may explain the agonistic activity of this peptide.
Abrogation of a putative intramolecular PPII-PRD
association [5] is a first line possibility in view of the fact
that the PRD contains a motif (β-turn/H4) in close contact
with the AF-2 region involved in recruitment of LxxLL-
containing coactivators [35]. Insofar as the deletion of the
P295-T311 sequence leads to a constitutive ERα-mediated
transcription [13], one may propose that this region impedes
the expression of the AF-2 domain in unliganded ER. Does
ERα17p act by itself on AF-2 topology like LxxLL-
containing coactivators, or by selectively favoring
recruitment of such coactivators, possibly via an exchange
process [54, 55]? This question to which no answer could be
provided so far is obviously worth of further investigations.
When given to MCF-7 cells, ERα17p induced responses
usually found under stimulation with estrogenic ligands
[13]. Studies reported here, conducted with a highly purified
hERα preparation, established that ERα17p associates with
the receptor at a site distinct from its ligand binding pocket.
Such a property led us to assume that the peptide may
bypass in the ERα transactivation mechanism the initial
step which is reflected by major conformational changes
required for AF-1 and AF-2 expression, associated/
subsequent recruitment of LxxLL-containing coregulators and
closing of the hormone binding pocket [54]. The finding
Fig. (9). Schematic representation of the potential mechanism by which ER 17p may activate ER . The model proposes a
repressive P295-T311 – H4 intramolecular interaction suitable for corepressor and/or chaperone binding ( ).By abrogating this
inactive conformation, E2 favors the recruitment of LxxLL-containing coactivators (⊕) at AF-2 which contributes to the closing of the
hormone binding pocket. ERα17p by interacting with the β-turn/H4 motif would either modify the AF-2 topology for coactivator
recruitment or play, by itself, the role of the coregulator. Concomitant release of bound corepressor is also indicated.

354 Letters in Drug Design & Discovery, 2007, Vol. 4, No. 5 Gallo et al.
The potential release of bound corepressor(s) and/or
chaperones may also be examined, since the PRD region was
recently shown to associate with Hsp70 [56]. In this context,
it should be stressed that ERα17p decreases the capacity of
the receptor to bind the hormone in whole cell assays, a
property which might result, at least in part, from the
dissociation of receptor-chaperone complexes. As yet, the
location of the ERα motif involved in ERα17p recognition
is still uncertain. Demonstration of a direct association in
solution of ERα17p with the β-turn/H4 region is of prime
importance to validate our statement (illustrated in Fig. (9)).
The synthesis of ERα17p analogs labeled with
photoactivable crosslinkers for irreversible attachment to the
receptor, followed by mass spectrometry-assisted protein
fragment analysis after enzymatic digestion seems to us an
appropriate strategy to achieve such a demonstration [57].
Isothermal titration calorimetry (ITC) or nuclear magnetic
resonance (NMR) experiments might be also used to uncover
ERα17p / ERα ligand binding domain interactions.
specificity of action. However, like most LxxLL-containing
peptide mimics (called now PERMs [67, 71, 74]), ERα17p
acts at relatively high concentration (IC50 ≈ 5 µM [13])
hardly appropriate for therapeutic applications. Besides, its
growth promoting activity on ERα-positive cells precludes
its potential use in women with high risk of breast cancer
development. Thus, ERα17p, as such, does not currently
appear as a promising drug candidate. Nevertheless, by
highlighting a novel ligand-independent ERα mode of
activation, it provides conceptual basis for the future
development of analogs capable of modulating ERα
transcriptional activity. From this point of view, docking
methodologies seem to us a first-line approach for the design
of such analog compounds.
ACKNOWLEDGEMENTS
This work has been supported by grants from the Belgian
Fund for Medical Scientific Research (Grant n° 3.4512.03),
the CGRI-CNRS/FNRS (Grant n° 18217), and the Fondation
MEDIC. D. Gallo is a recipient of a grant from the Fonds
Jean-Claude Heuson. G. Laurent is Senior Research
Associate of the National Fund for Scientific Research
(Belgium). We thank E. Arnould for her active contribution
in this work and J. Richard for her excellent secretarial and
editorial assistance. We are grateful to Drs. S. Fermandjian,
L. Zargarian (CNRS-UMR 8113, Institut Gustave Roussy,
Villejuif) and N. Goasdoué (CNRS-UMR 7613, Université
Pierre et Marie Curie, Paris) for circular dichroism
experiments. We are also indebted to T. Verjat and B.
Mougin (BioMérieux; Lyon, France) for NASBA analysis.
On a physiological point of view, exchange between
corepressors and coactivators at ERα level is a dynamic and
cyclic mechanism which cannot simply be accounted for by
stochastic Brownian motion [58, 59]. On the other hand,
intracellular Ca2+ signaling is known to entail pulses of
Ca2+ concentrations. Does CaM, a major sensor of Ca2+
intracellular oscillations, play a role in coregulator exchange?
ERα17p, which contains the receptor CaM binding site, was
initially synthesized with the aim to assess the contribution
of CaM in ERα transactivation [13]. As yet, this association
has only been shown to stabilize ERα [11, 60, 61] and
enhance its binding to DNA [62, 63]. Data reported here
suggest that CaM, by interacting with the P295-T311
platform, may contribute to orchestrate coregulator exchange
process (i.e. Hsp70 release in favor of the recruitment of
p300 coactivator family members [56]). In this view one
may assume that Ca2+ oscillations, like a metronome, may
modulate the cyclic profile of the ERα transactivation
mechanism.
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