Báo cáo khoa học: G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid pdf

G protein-coupled receptor 30 down-regulates cofactor expression
and interferes with the transcriptional activity of glucocorticoid
Timo Ylikomi
1,2
, Annika Vienonen
1
and Tytti M. Ahola
1
1
Department of Cell Biology, Medical School, 33014 University of Tampere, Finland;
2
Department of Clinical Chemistry,
Tampere University Hospital, Tampere, Finland
G protein-coupled receptor 30 (GPR30) has previously been
described to b e important in s teroid-mediated growth and to
inhibit cell proliferation. Here we investigated whether the
effect of GPR30 on cell growth is dependent on steroid
hormone receptors. We stably introduced GPR30 in
immortalized normal mammary epithelial (HME) ce lls using
retroviruses for gene delivery. GPR30 inhibited the growth
and proliferation of the cells. They e xpressed glucocorticoid
receptor, but not estrogen or progesterone receptor. GPR30
down-regulated the expression of cofactor transcription
intermediary factor 2 (TIF2) analyzed using quantitative
RT-PCR analysis, and also diminished th e expression of
TIF2 at protein level analyzed by Western blotting using
nuclear extracts from mammary epithelial cells. W hen HME
cells were transiently transfected with the glucocorticoid
response element MM TV-luc reporter p lasmid, stable
expression of GPR30 r esulted in the abolition of ligand-
induced transactivation of t he promoter. I n COS cells,

transient transfection of GPR30 with glucocorticoid recep-
tor a resulted in an abrogation of the MMTV-luc and GRE-
luc r eporter activities induced by dexamethasone. The results
suggest a novel mechanism by which membrane-initiated
signaling i nterferes with steroid signaling.
Keywords: cofactor; glucocorticoid; G PR; proliferation;
transcription.
Glucocorticoids play a vital role throughout physiology.
The physiological response a nd sensitivity t o glucoco rticoids
varies among species, individuals an d cell types and duri ng
the cell cycle [1,2]. Moreover , several pathological condi-
tions are a result of glucocorticoid resistance [3]. The
molecular basis of glucocorticoid resistance, however, varies
widely and is not completely understood.
Glucocorticoids b elong to a member of the steroid
hormone family which bind to receptors triggering their
conformational change and nuclear translocation. The
hormone–receptor complex binds to specific response
elements in target genes and interacts with several coacti-
vators, including steroid receptor c oactivator 1 (SRC-1)
family [4].
SRC-1 coactivators such as transcription intermediary
factor 2 (TIF2) are able to enhance transcriptional activa-
tion by the receptor via mechanisms that include recruit-
ment of the general coac tivator, cAMP-response element
binding protein-binding protein (CBP), and histone acety-
lation [5]. TIF2 coactivates all steroid, t hyroid, retinoid a nd
vitamin D receptors [6,7]. It i s widely e xpressed in different
human tissues, implying an important role for TIF2 in cell
function [6]. Two reports have addressed its role in growth

regulation. Its mouse homologue, glucocorticoid receptor
interacting protein-1 (GRIP-1), is critical for skeletal muscle
differentiation in the mouse [8]. The reduction of TIF2
expression by antisense oligodeoxynucleotides inhibits lig-
and-stimulated ER transcriptional activity and DNA syn-
thesis in MCF-7 cells, this constituting evidence of the role
of TIF2 in growth stimulation [9].
As coactivators function as transcription a mplifiers,
changes i n their expression levels would markedly alter
receptor-mediated transcriptional activity. There is some
evidence on the r egulation of c ofactors. A potential pathway
for TIF2 regulation was suggested by Borud and associates.
In transient transfection assays, the function and protein
level of over-expressed TIF2 was impaired by activation
of protein kinase A (PKA) [10]. Indeed, TIF2-mediated
coactivation of the nuclear receptor peroxisome prolifera-
tor-activated receptor, liver X nuclear receptor and retinoid
X receptor was repressed by PKA catalytic subunit over-
expression. Steroid hormones regulate SRC-1 expression in
Correspondence to T. M. Ahola, Medical School, 33014 University of
Tampere, Finland, Fax: +358 3 215 6170; Tel.: + 358 3 215 8942;
E-mail: Tytti.Ahola@uta.fi
Abbreviations: BPE, bovine pituitary extract; BrdU, bromodeoxyuri-
dine; CBP, cAMP-response element binding protein-binding protein;
CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein;
ER, estrogen receptor; GDI, guanine nucleotide dissociation;
GR, glucocorticoid receptor; GPR, G protein-coupled receptor;
GRE, glucocorticoid response element; GRIP-1, glucocorticoid
receptor interacting protein-1; hrEGF, human recombinant epidermal
growth factor; HME, human normal mammary epithelial cells;

hTERT, human telomerase reverse transcriptase subunit;
IL, interleukin; MAPK, mitogen-activated protein kinase;
MMTV, murine mammary tumor virus; PR, progestrone receptor;
PI3-K, phosphatidylinositol 3-kinase; PKA, protein kinase A;
SUMO-1, small ubiquitin-related modifier-1BrdU, bromodeoxy-
uridine; SRC-1, steroid receptor coactivator 1; TBP, TATA binding
protein; TIF2, transcription intermediary factor-2.
(Received 1 7 March 2004, revised 13 August 2004,
accepted 2 September 2004)
Eur. J. Biochem. 271, 4159–4168 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04353.x
rats. Dexamethasone [4] and estrogen down-regulate, and
thyroid hormone up-regulates SRC-1 cofactor expression in
rat tissues [11].
The regulation of GR activity by growth factors and
cAMP is well established, and in most cases req uires the
presence of gluco corticoid. T he role of GPR in gluco-
corticoid-mediated signaling w as suggested b y Schmidt
and coworkers [12]. b
2
-adrenergic receptors were able to
modulate GR transactivation by stimulating phosphati-
dylinositol 3-kinase (PI3-K) [12]. In general, GPR signals
a large variety of stimuli g enerated by different hor-
mones, neurotransmitters, sensory stimuli and odorants
[13]. The GPR superfamily numbers close t o 2000 G
protein-coupled receptors [14,15], with a major contribu-
tion in the growth regulation of different normal and
cancer cells. The major e vidence to date concerns the role
of GPR in enhancing cell proliferation [16–20]. There are
also examples suggesting t he antiproliferative activity of

GPRs [21–23].
The role of GPR30 in glucocorticoid-mediated signaling
has not hitherto been described . Many groups indep end-
ently demonstrated GPR30 in different tissues in the late
1990s, and confirmed its ubiquitous expression pattern.
Based on the amino acid sequence of GPR30, it was
identified as an orphan transmembrane receptor bearing
some degree of similarity to chemokine receptors such as
interleukin (IL) 8 and angiotensin II [24]. GPR30 belongs to
the rhodopsin-like peptide receptor family, and the chemo-
kine receptor-like 2 subfamily, w hich also includes as its
members GPR41 (lung; rat) and a receptor similar to
GPR30 (uterus, leiomyosarcoma; human). GPR30 is pref-
erentially expressed in ER-positive breast cancer cells as well
as in endocrine tissues, but also, e.g. in endothelial cells,
lung, heart, lymphoid tissues/cells and in the central nervous
system [24–28]. The gene is shown t o be up-regulated by
fluid shear stress [25], p rogestins and progesterone [29].
GPR30 inhibits proliferation of MCF-7 b reast cancer cells
[30], and its close homologue in the rat induces apoptosis
through the p53-pathway [31].
It has been suggested t hat GPR30 plays a critical role in
progestin- as well as estrogen-mediated signaling [30,32,33].
Here we sought to establish whether GPR30 inhibits growth
by affecting steroid receptor activity or through other
cell signaling pathways. We thus investigated the effect of
GPR30 on the growth an d ste roid receptor activity of
immortalized mammary epithelial cells that expressed GR
but not estro gen receptors (ER) or progesterone receptors
(PR). Unprecedentedly, GPR30 down-regulated the expres-

sion o f cofactor TIF2, interfered with the transcriptional
activity of GR and inhibited growth. This observation
suggests a mechanism by which membrane-initiated signa-
ling interferes with steroid signaling.
Experimental procedures
Hormones and growth factors
17b-Estradiol, medroxyprogesteroneacetate, dexametha-
sone, human recombinant epidermal growth factor
(hrEGF) and hydrocortisone were obtained from Sigma
Chemical Co. Mifepristone (RU486) was a gift from
Roussel Uclaf (Paris, France). Insulin, amphotericin-B
and bovine pituitary extract (BPE) were purchased from
Gibco BRL (Paisley, UK).
Cell culture
Packaging cell line PT67 and NIH373 cells were cultured in
Dulbecco’s modified Eagle’s medium containing 10% fetal
bovine serum, 100 UÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin (Gibco BRL ). hTERT-HME1, a human
primary e pithelial cell line stably e xpressing the human
telomerase reverse transcriptase subunit (hTERT) was
obtained from Clontech and had undergone 134.74 popu-
lation doublings at the beginning of the experiment. The cell
line was maintained in mammary epithelial basal medium
supplemented with 52 lgÆmL
)1
BPE, 0.5 lgÆmL
)1

hydro-
cortisone, 10 ngÆmL
)1
hrEGF, 5 lgÆmL insulin, 50 lgÆmL
Gentamicin (Gibco BRL) and 5 0 n gÆmL
)1
Amphotericin-B.
COS cells were maintained in Dulbecco’s modified Eagle’s
medium/nutrient mixture F-12 supplemented with 5 % fetal
bovine s erum and p enicillin/stre ptomycin. In transient
transfection assays, fetal bovine serum was replaced by
10% dextran-coated, charcoal-stripped fetal bovine serum.
RNA isolation
RNA w as isolated from cells using the RNAqueous
TM
kit
(Ambion). Cells were harvested in t rypsin/EDTA a nd
washed with NaCl/P
i.
The c ell pellet w as mixed w ith
900 lL Lysis/Binding solution from th e kit. RNA isolation
was carried out according to manufacturer’s instructions.
Cell growth assay
Cells were seeded in 96-well plates at a density of 1 · 10
3
cells per well in the experimental medium, and were allowed
to attach for 1 day. Relative cell numbers were measured
using the crystal violet method [34]. The cells were fixated
and stained with crystal violet, and d ried cells were diluted
with acetic acid. A bsorbance was measured at a 590 n m

using a Victor 1420 Multilabel counter (Wallac, Turku,
Finland).
Cell proliferation assay
This assay was carried out as described previously using
immunohistochemistry [30]. In brief, cells were plated on
glass slides. Bromodeoxyuridine (BrdU) was added to the
growth medium, and BrdU was visualized using mono-
clonal anti-BrdU Ig (Sigma). Alternatively, the proliferation
index was measured using anti-(Ki-67) Ig (Sigma).
Establishing retrovirus-producing cell lines
GPCR-Br/GPR30 was kindly provided by D. Thompson,
Department of Surgery, Standford U niversity, CA, USA
[24]. GPR30 was cloned i n the BamHI and NotIsitesofthe
pLEGFP-N1 vector (Clontech) replacing enhanced green
fluorescent protein (EGFP). In order to create a pL-N1
control vector, restriction sites were filled using Klenow
Fragment (MBI Fermentas, Hanover, MD), and the
product was blunt-ligated with itself using 300 U T4 ligase
(MBI Fermentas). Fusion o f GPR30 to EGFP was effected
4160 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
in pLEGFP-N1 vector at SalIandBamHI sites. The
following primers were used for cloning at the pLEGFP-N1
vector: GPR30-forward 5¢-TAATAAGTCGACGGGTC
TCTTCCT-3¢ and GPR30-reverse 5¢-ATTATTGGATC
CTACACGGCACTGC-3¢. V iruses capable of i ntroducing
pLEGFP-N1, pLEGFP-N1/GPR30, pL-N1 and pL-N1/
GPR30 vectors were established in the PT67 packaging cell
line derived from ATCC (American Type Culture Collec-
tion, Manassas, VA, USA). PT67 cells (5 · 10
4

)were
transfected using 1 lL lipofectamine 2000 ( Gibco BRL) and
1 lg pla smid for 24 h. After 36 h incubation with the
growth medium, cells were grown in the presence of
800 lgÆmL
)1
Geneticin (Sigma) for 1 week to select those
containing pLEGFP-N1, pL-N1 and pL-N1/GPR30 vec-
tors. Six colonies were isolated from each plasmid, and a
strain showing the highest viral titer was selected for further
studies. Viral titer was determined using NIH373 cells as
recommended i n the Retroviral Gene Transfer and Expres-
sion User Manual (GEU manual, Clontech). PT67 cells,
which stably (pLEGFP-N1, pL-N1, pL-N1/GPR30) or
transiently (pLEGFP-N1/GPR30) produced viruses, were
grown for 4–5 days in the medium, and the viral titer
determined as recommended in the GEU manual (Clon-
tech) before storage at )80 °C.
Cell infection
In preliminary studies, optimal infection conditions were
determined, and 13 viruses per cell were u sed to infect
hTERT-HME1 cells. The cells were infected at two separate
times, 12–24 h between infections. Polybrene 8 lgÆmL
)1
(Sigma Chemical Co.) was added to reduce charge repul-
sion in some infections. After 72 h incubation with the
growth med ium, the cells were grown in the presence of
800 lgÆmL
)1
Geneticin (Sigma Chemical Co.) to select

those containing pLEGFP-N1, pLEGFP-N1/GPR30,
pL-N1 and pL-N1/GPR30 vectors.
Enhanced green fluorescent protein measurement
Cells were plated on 96-well plates as in the cell growth a ssay.
At indicated time points the medium was removed an d 20 lL
Cell Culture Lysis Reagent (Promega, Madison) was added
to the wells. The plates were shaken for 1 5 min at 500 r.p.m.
Fluorescence was measured using a Victor 1420 Multilabel
counter (Wallac) at wavelengths 485/535 nm. In order to
correlate absorbance with protein concentration, a standard
curve was made using recombinant EGF protein (Clontech).
RT-PCR analysis
In order to measure the regulation of mRNA, one-step
RT-PCR was p erformed. A LightCycler instrument ( Roche,
Mannheim, Germany) a nd LightCycler RNA Master SYBR
Green I kit (Roche) were u sed in the a ssay. Specific primers
for GPR30 and house-keeping gene TATA binding protein
(TBP) were used as described previously [29]. To measure
TIF2 exp ression, forward p rimer 5¢-ATCTCCAAGGCAA
GATCA-3¢ and reverse prime r 5 ¢-GTGCCATCAGA
CAAGGAA-3¢ were used. Primer pair 1 resulted in a
PCR product of 216 bp. Additionally, forward primer
5¢-GAGCCCC AAGAAGAAAGA-3¢ and reverse primer
5¢- CATCCAAAATCTCC TCCA-3¢ were used. Primer pair
2 resulted in a PCR product of 230 bp. The forward and
reverse primers were designed in different exons. The reverse
transcription was performed at 6 1 °C for 20 min and
denaturation at 95 °C for 30 s. Forty-two cycles of PCR
were carried out. The cycle included d enaturation at 95 °C
for 1 s, annealing at 51 °C for 5 s and elongation at 72 °Cfor

10 s. The melting curve was obtained as described elsewhere
[29]. The final results, expressed a s N-fold differences in gene
expression between the GPR30-transfected and vector-
transfected samples, were arrived a t as f ollows:
N
gene
¼ðTIF2
GPR30-transfected
=TBP
GPR30-transfected
Þ=
ðTIF2
vector-transfected
=TBP
vector-transfected
Þ:
The relative concentrations used for expression difference
calculations were obtained from the calibration curves.
Concentration v alues for TI F2 were taken from the
calibration curve made from serial dilutions (500 fi
100 fi 20 fi 4 ng) of the total RNA.
Immunoblotting
Cells were harvested by a cell scraper and calculated in a
Burker cell chamber. Nuclear extracts were prepared from 2
to 10 · 10
6
hTERT-HME cells as described previously [35].
Protein concentrations were determined using BCA Protein
Assay Reagent (Pierce, Rockford, IL, USA). Immunoblot-
ting was carried out as previously described [36]. Nuclear

extracts or cell pellets were mixed with 2· SDS-sample buffer
and boiled for 5 min. Equal amounts of nuclear proteins
(196 lg) or cells (3 ·10
5
) of cell lysates were resolved in 12%
polyacrylamide g el and transferred t o a nitrocellulose
membrane with an electrophoretic transfer apparatus. After
blocking, the membranes were incubated o vernight at 4 °C
with GRIP-1 (F-20) antibody at a final concentration of
1 lgÆmL
)1
(Santa Cruz Biotechnology, Santa Cruz, CA,
USA). Biotinylated anti-goat IgG (Vector Laboratories I nc.,
Burlingame, CA) and horseradish peroxidase avidin D
(Vector Laboratories Inc.) were used as secondary antibod-
ies. Alternatively, membranes were incubated with anti-GR
(P20) (Santa Cruz Biotechnology), NCL-L-PR (Novocas-
tra), NCL-ER-6F11 (Novocastra) or anti-b-actin (Sigma).
Peroxidase-conjugated goat anti-rabbit I gG (Cappel, West
Chester, PA, USA) for GR and peroxidase-conjugated goat
anti-mouse IgG (Cappel) for PR, ER and b-actin were used
as secondary antibodies. After washing, labeled proteins
were detected by enhanced chemiluminescene.
Transient transfection assays
Cells we re transfected using the Lipofectamin 2000 method
as recommended ( Gibco B RL). A DNA mixture of
pCMVbGal 50 ng, pSG5 or GRa (2.5 lg), pBk-CMV or
GPR30/pBk-CMV (0.1, 0 .3, 0.6 lg), G RE-tk-luc ( 2.5 lg) or
MMTV-tk-luc (2.5 lg)wasusedtotransfectCOScells;and
aDNAmixtureofpCMVbGal ( 50 ng), GRE-tk-luc

(2.5 lg) or MMTV-tk-luc (2.5 lg)wasusedtotransfect
hTERT-HME cells. Transfection was carried out for 6 h for
COS cells and 24 h for hTERT-HME cells. A fter transfec-
tion, the cells were incubated for 4 h with the basic medium
lacking g lucocorticoid. Luciferase activity in the samples was
Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4161
measured 48 h a fter glucocorticoid addition applying the
Luciferase Assay System as recommended (Promega) and
using a 1450 Microbeta Plus Liquid Scintillation Counter
(Wallac). Equal transfection efficiency was confirmed by
measuring b-galactosidase activity in heat-treated (10 m in
at 50 °C)lysatesasrecommendedintheb-Galactosidase
Enzyme Assay system (Promega), with a Victor 1420
Multilabel c ounter at a wavelength of 450 nm (Wallac).
Results
Mammary epithelial cells expressed GR
We characterized the steroid receptor composition of H ME
cells. T he cells expressed G R as a ssessed using immuno-
blotting analysis (Fig. 1). No PR or ER a was d etected in the
cells. MCF-7 cells were used as a positive control.
GPR30 inhibited proliferation of mammary epithelial
cells
GPR30 has a critical role in progestin- and estrogen-
mediated signaling [30,32,33]. It also inhibits proliferation of
ER- and PR-positive MCF-7 cells [30]. We investigated
whether the effect of GPR30 on growth is dependent on
steroid receptors and whether GPR30 inhibits the growth of
non-neoplastic breast epithelial cells which did not express
ER or PR. We stably introduced GPR30 to commercial
HME cells immortalized by hTERT, using retroviral-

mediated gene delivery. The immortalized HME c ells were
cultured without serum, thus avoiding the influence of
unknown growth factors. As a control we used cells infected
with an empty vector pL-N1. GPR30 mRNA was expressed
in HME cells 28-fold compared to control cells as measured
by quantitative PCR 96 h after plating (Fig. 2).
Interestingly, when relative cell growth was measured,
growth was inhibited by 23–34% between 48 and 168 h
(Fig. 3A). Corresponding results were obtained from two
separate infections. Time point zero taken to be the point
when cells were attached to plates, this usually taking 1 day.
To further study the mechanism whereby GPR30 regu-
lates growth in these cells, we measured the cell proliferation
index using a BrdU incorporation assay and KI-67 immu-
nostaining. Before the BrdU e xperiment, the cells were
allowed to attach, for 1–2 days, the medium was c hanged
and 0 h time point measured. BrdU incorporation a nalysis
revealed that proliferation was inhibited by 60–87%
between 0 and 72 h in H ME cells (Fig. 3B). Cell prolifer-
ation was also measured using KI-67 as an indicator of cells
in cycle. Prior to the KI-67 assay, HME cells were arrested
at the G
0
/G
1
phase by growth factor deprivation (mammary
epithelial basal medium present without supplements) for
24 h. Ki-67 staining revealed that GPR30 inhibited prolif-
eration by 40% at 2 h and inhibition increased to 53% 12 h
after growth factor deprivation (Fig. 3C).

A
B
C
D
β-actin
GR
ER
PRB
MCF-7 HME
PRA
Fig. 1. HME c ells e xpr ess G R. The receptor composition of immor-
talized HME cells was characterized using immunoblotting. Cell
lysates o f MCF-7 cells (used as positive control) and HME cells
transfected with empty pL-N1 vector w ere run in po lyacrylamide gel.
Proteins were transferred to the nitrocellulose membrane and detected
with the antibodies against PR A and B (A), ERa (B) an d GR (C). T o
confirm equal loading efficiency t he m em branes were incu bated with
anti-b-actin (D).
50
*
40
30
20
10
0
C GPR30
GPR30 mRNA
fold induction
Fig. 2. HME cells express GPR30. Stable expression of GPR30 in
HME cells was achieved u sing retroviral-mediated g ene delivery. The

relative expression of GPR30 mRNA was stu died u sing q uantitative
RT-PCR analysis. Total RNA was extracted from cells grown to
50–70% confluence in thre e independe nt experiments. T he result is
presented as mean of three experiments. Statistical significance was
calculated using Student’s paired t-test. Differences in expression were
considered significant at *P < 0.05.
4162 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
GPR30 down-regulated cofactor expression
in hTERT-HME cells
GR affects gene transcription by a mechanism which is
enhanced by cofactors. We therefore characterized the effect
of GPR30 on the expression of receptors and cofactors. I n
our preliminary analysis, GPR30 affected the expression
level of TIF2 mRNA, but not of other cofactors (nuclear
receptor corepressor, silencing mediator of retinoic acid and
thyroid hormone receptor, p300/CBP-associated factor,
Ras-related C3 botulinum toxin substrate 3) or receptors
(GR, androgen receptor, PR, ER) studied. We thus verified
the effect of GPR30 o n the expression level o f TIF2 in HME
cells using quantitative RT-PCR analysis a nd two different
primer pairs. Expression of TIF2 was down-regulated by
5
C
A
B
C
GPR30
*
**
***

*
*
*
**
*
*
*
**
*
*
C
GPR30
C
GPR30
4
3
2
1
0
0 24 48 72 96 120 144 168
Time (h)
40
30
20
10
0
024487296
Time (h)
20
25

15
5
10
0
0246810
12
14
Time (h)
KI-67%
BrdU%
Relative Cell Number
(A 590 nm)
Fig. 3. GPR30 inhibits proliferation of HME cells. (A) Cells were
plated on 96-well plates and the relative cell number was measured
from HME cells transfected either with GPR30 or empty vector using
crystal violet staining. (B) Cell proliferation was measured u sing the
bromodeoxyuridine (BrdU) incorporation assay, and BrdU-incor-
porated cells were visualized by immunostaining with anti-BrdU Ig at
indicated time points. (C) Cells were arrested at the G
1
phase by
growth factor deprivation. Thereafter, the cells were cultured with
growth medium and immunostained with the a ntibody against Ki-67
at indicated time points. The percentage of Ki-67-positive cells was
counted. The data presented are the means of three to four replicates.
Statistical significance was calculated using the t-test as indicated in
Fig. 2. C, control.
100
80
60

40
20
A
B
C
0
C GPR30
**
*
100
80
60
40
20
0
C GPR30
C GPR30
TIF-2 mRNA
% of control
TIF-2
TIF-2 mRNA
% of control
Fig. 4. GPR30 d own-regulates the expression of TIF2 protein. The
regulation of cofactor was studied in HME cells stably expressing
GPR30. The relative expression of TIF2 mRNA was stud ied using
quantitative RT-PCR analysis and two different primer pairs: (A)
primer pair 1 and (B) primer pair 2 . W hen c ells were grown to 5 0–70%
confluence, total RNA was extracted for analysis. Results are pre-
sented as means of three replicates. (C) TIF-2 protein expression was
studied the nuclear extracts of HME cells (196 lg) using immuno -

blotting. C, control.
Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4163
90% a nd 40% by GPR30 a s a nalyzed u sing RT-PCR
analysis and two different primer pairs (Fig. 4A,B).
To study the c ofactor regulation at p rotein level, we used
specific antibodies against cofactor TIF2. In immunoblot-
ting analysis GPR30 down-regulated TIF2 expression in
HME nuclear extracts (Fig. 4C).
GPR30 inhibited transcriptional activity of the
glucocorticoid response element in hTERT-HME cells
The finding that GPR30 down-regulated the expression of
cofactor TIF2 led us to study whether GPR30 affects the
transcriptional activation o f the glucocorticoid response
element in HME cells which expressed endogenous GR
receptor but not PR or ER. We used HME cells infected
with GPR30 or pL-N1 control v ector. We transiently
transfected the cells with MMTV-luc reporter gene, which
contains glucocorticoid response elements. In control cells,
MMTV promoter was stimulated 370% by hydrocortisone
treatment (Fig. 5A). In GPR30 stable e xpressing cells, the
effect of hydrocortisone on MMTV-luc activity w as almost
abrogated. Without the presence of hydrocortison, GPR30
had no effect on the activity, indicating that the effect is
glucocorticoid-dependent. Equal transfection efficiency was
confirmed using a CMV-bgal vector as marker for trans-
fection efficiency.
GPR30 inhibited the transcriptional activity
of the glucocorticoid receptor in COS cells
To confirm that the effect of GPR30 on transcriptional
activity was mediated through GR, we studied the phe-

nomenon in steroid receptor-negative COS cells using the
transient transfection approach. COS ce lls were transfected
with the expression vectors encoding GRa and GPR30 (or
with the corresponding control vectors) together with the
reporter construct MMTV-tk-luc. MMTV-tk-luc promoter
activity was enhanced 730% by dexamethasone treatment
in control cells (Fig. 5B). When the cells were transfected
with GPR30 and treated with dexamethasone, the luciferase
activity was almost abolished. GPR30 did not affect the
target promoter in the absence of dexamethasone, confirm-
ing that the effect is glucocorticoid-dependent.
To study whether the effect of GPR30 on MMTV
activity was c oncentration-dependent, we t ransfected COS
cells with increasing amounts of GPR30 plasmid. The
400
AB
CD
350
**
**
**
*
300
250
200
150
Luciferase Activity
% of control
Luciferase Activity
% of control

Luciferase Activity
% of control
Luciferase Activity
% of control
100
400
350
300
250
200
150
100
400
500
600
700
800
900
1000
300
200
100
400
500
600
700
300
200
100
GPR30

––
–+–
+
++
–
––
–
++
+
++
+++
COR
GPR30
GR
DEx
–
––
–
++
+
++
+++
GPR30
GR
DEx
–
–
–
+
+

++
+
+
+
++
GPR30 0.1 0.3 0.6
GR
DEx
Fig. 5. GPR30 i mpairs the ability o f glucocorticoid to a ctivate transcription. (A) H ME ce lls stab ly ex pressing GPR30 (or control vector p LN-1) w ere
transfected with a m urine mammary t umor virus-tk-luc reporter construct, and treated with hydrocortisone. (B) COS cells were transfected with
MMTV-tk-luc reporter gene, and the expressio n vectors g luc ocorticoid receptor a and G PR30. The cells were treated with dex amethaso ne to ind uce
reporter gene transcription. (C) COS cells were transfected w ith MMTV-tk-luc, glucocorticoid receptor a and increasing c onc entrations of GPR30.
(D) COS cells were transfected with a glucocorticoid response element-tk-luc reporter construct, and expression vectors containing glucocorticoid
receptor a and GPR30. Luciferase assays were p erformed 48 h after addition of dexamethasone. The figure shows the mean v alue of tr iplicate
transfections. DEX, dexamethaso ne ; GR, glucocorticoid receptor.
4164 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
increase in GPR30 expression was f ound to inhibit t he
luciferase activity in a concentration-dependent manner
(Fig. 5 C).
When COS cells were transfected with the other gluco-
corticoid response element, GRE-tk-luc, dexamethasone
induced an increase in GRE-tk -luc reporter activity by
360%. Similarly, when the c ells were transfe cted with
GPR30 and treated w ith dexamethasone, t he luciferase
activity was almost abolished (Fig. 5D). GPR30 by itself
had n o effect on transcriptional activation of GR-tk-luc.
Equal transfection efficiency was confirmed using a CMV-
bgal v ector as marker for transfectio n efficiency.
GPR30 fusion protein inhibits growth and cofactor
expression

We have previously established changes in GPR30 expres-
sion level only at the RNA level. In order to confirm t hat the
effect of GPR30 on the regulation of cofactor and g rowth
was due to GPR30 protein expression, we measured the
effects of GPR30 fused with EGFP. As a control, we used
HME cells infected with the plasmid-expressing EGFP.
Fluorescence was measured in the cell lysates at a
wavelength of 485/535 nm. E GFP was expressed in control
cells (Fig. 6 A). GPR30-EGFP expression was detected in
experimental cells at a high level.
We also measured relative cell growth u sing crystal v iolet
staining. Cell proliferation was inhibited by GPR30-fusion
protein when comparison was made with cells expressing
EGFP or parental HME cells (Fig. 6 B). In accord with
previous results, TIF2 protein levels were down-regulated
by GPR30-fusion protein analyzed by Western b lotting
(Fig. 6C). The re sult suggests that the measured effects on
growth and the expression of cofactor were due to GPR30
protein expression.
Discussion
Molecular discrimination of cells differing in their sensitivity
to steroids is essential in diseases involving resistance to
steroid h ormone treatment. In the present study, we
addressed the question of the ability of GPR30 to regulate
steroid hormone-induced transcription, particularly as a
critical role of GPR30 in steroid-mediated signaling has
been described [30,32]. We found that stable expression of
transmembrane receptor GPR30 in human immortalized
breast cells down-regulated the expression of cofactor TIF2,
resulted in abrogation of the transcription activity of GR

and inhibited c ell p roliferation. These r esults suggest a new
mechanism underlying glucocorticoid resistance. The data
also point to cross-talk with G proteins and steroid receptor
activity, and provide a mechanism whereby membrane-
initiated signaling might diminish cell proliferation.
Interestingly, GPR30 inhibited GR-mediated activation
of transcription both in non-neoplastic breast epithelial cells
and i n COS cells. Other studies have also indicated the role
of cytoplasmic s ignaling in s teroid hormone-mediated
transcription. b
2
-Adrenergic receptor stimulates GR trans-
activation by activating PI3-K [12], and Rho GDP-disso-
ciation inhibitor (GDI) modulates ER transcriptional
enhancement [36]. Additionally, the activation of PKA is
known to lead to s teroid receptor-induced transcription [10].
Inhibition of steroid hormone-induced trans cription by
cytoplasmic signaling has not, however, previously been
described.
We further sought to identify the mechanism by which
GPR30 would interfere with GR mediated activation of
transcription. We established that GPR30 down-regulated
the expression of cofactor TIF2. The crucial r ole of
cofactors in the activation of transcription has b een shown
in a number of studies. Indeed, over-expression of cofactors
HME
GPR30
pLEGFP-N1
HME
GPR30

pLEGFP-N1
pLEGFP-N1
GRP30
*
*
*
**
0.01
1.0×10
–04
1.0×10
–06
1.0×10
–08
0244872
Time (h)
rEGFP protein
mg/ml
Relative cell number
(A590 nm)
96 120
144
0
0
1
2
3
4
B
C

A
24 48 72
TIF-2
Time (h)
96 120
14
4
Fig. 6. Fusion protein of GPR30 inhibits proliferation and cofactor
expression. GPR30 was fused to EGFP. Retrovirus infection was used
to obtain stable expression of this construct in HME cells. Cells stably
expressing EGFP were used as control. (A) The amount of GPR30
fusion protein and EGFP in control cells was calculated by measuring
fluorescence at wavelengths 485/535. A standard curve was made f rom
EGFP recombinant prote in in order to correlate absorbance with
protein concentration . R esults ar e p rese nted as means of t hree re pli-
cates. (B) Relative c ell g rowth w as measured in GPR30-expressing and
control cells using crystal viole t staining. Ab sorbance was m easured at
a wavelength o f 590 nm (C) T IF2 protein expression in the nuclear
extracts of HME cells (196 lg) was de tected using imm uno blotting .
Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4165
enhances steroid hormone-induced transcriptional activity
[5–7], and a r eduction in cofactor TIF2 expression prevents
ERa-induced and ligand-induced activation of the promo-
ter 3xERE-TATA [9]. Differential expression of cofactors
has been proposed in explanation of some of the differential
effects of s teroid hormones. The m echanism whereby
GPR30 modulates TIF2 expression is not known. We were
not able to establish MAPK inactivation or activation by
GPR30 in HME cells, although MAPK activation/inacti-
vation has been established i n breast cancer cells [32,37].

Other studies have also established t he regulation of
cofactor activity by membrane-initiated signaling. Indeed,
regulation of GRIP1 and CBP coactivator activity by
cytoplasmic Rho GDP-GDI leads to ER activation [36].
Our study therefore suggests that the down-regulation of
TIF2 expression by GPR30 i s a mechanism involved in the
establishment of steroid hormone resistance by GPR30.
GPR30 most probably a lso induces glucocorticoid
resistance by mechanisms other than down-regulation of
TIF2 expression. Ligand binding is the main inducer of the
transcriptional activity, but post-translational modifications
have also been shown to play an i mportant role. GR activity
is regulated through receptor phosphorylation, e.g. by the
PKA and MAPK pathway [38] and through the covalent
addition of the small ubiquitin-related modifier-1 (SUMO-
1) peptide [39]. Interestingly, GPR30 has been proposed to
activate the PKA p athway in breast cancer cells [33].
Activation of the MAPK pathway has been shown to affect
the transcriptional activation o f various receptors by
modulating cofactor recruitment [40] and affecting receptor
degradation [ 41,42]. I t has also been shown that GPR30
regulates MAPK activity [32,33,37].
In addition to interfering w ith the transcriptio nal activity
of GR, GPR30 was seen here to reduce the growth and
proliferation of immortalized breast cells. There was a more
profound effect on cell proliferation than on cell number, a
phenomenon previously described in the case of mutant
activated Ga-proteins. Ga inhibits the ability of MCF-7
cells to proliferate and to form tumors in athym ic mice [43].
Additionally, adenovirus-directed expression of ac tivated

mutant Ga inhibits the growth of e stablished tumors b y
inhibiting the M APK pathway [44]. W e have shown
previously that GPR30 is a ble to i nduce G
0
/G
1
phase arrest
in MCF-7 breast cancer cells [30]. Thus our result suggests a
molecule whose growth effect is independent of the differ-
entiation status (normal vs. c ancer) of the epithelial c ells in
the mammary gland.
We also show here that the abolition of hormone-
mediated transactivation of a GRE- responsive element by
GPR30 is associated with growth inhibition in HME c ells.
Down-regulation of TIF2 expression and the resultant
glucocorticoid insensitivity m ight contribute to the growth-
inhibitory effects of GPR30 in these cells, because gluco-
corticoid has been shown t o inhibit growth in b reast cancer
cells [45]. Such a conception is suppo rted by the findings of
Cavarretta and associates, who showed that a reduction in
TIF2 expression was a ble to p revent estrogen-induced
proliferation in MCF-7 cells [9].
We h ave also shown previously that GPR30-regulated
growth was independent of the presence of steroid hor-
mones [30]. Thus it can be concluded that the ligand for
GPR30 is likely to be in the BPE extract or be secreted into
the medium by mammary epithelial cells. Computer
experiments suggest that the hydrophilic cyclopeptide
derived from alpha-fetoprotein interacts with GPR30 and
may execute its action by interaction with GPR30 [46].

Interestingly, human alpha-fetoprotein peptides also bind s
to the estrogen receptor and estradiol, and suppresses breast
cancer [47]. In th e adult, only trace amounts o f alpha-
fetoprotein is detected [48], but in a pathological state in
adult life, alpha-fetop rotein levels rise (e.g. hepatic tumor).
Thus the ligan d of G PR30 might be secreted into the
medium by breast cancer and immortalized mammary
epithelial cells.
In conclusion, our results imply that GPR30 interferes
with glucocorticoid-mediated transcriptional activation
associated with down-regulation of cofactor TIF2 expres-
sion and growth inhibition. Glucocorticoids have a critical
role in the regulation o f human body functions, and
constitute widely used drugs in medicine. The physiological
response and sensitivity to glucocorticoids varies [1–3], but
the m olecular basis of glucocorticoid r esistance is not
completely understood. Thus our result suggests a novel
mechanism by which membrane-initiated signaling inter-
feres with growth and glucocorticoid responsiveness.
Acknowledgements
Plasmid GPCR-Br/GPR30 was provided by Dr D. Thompson. We
thank Heimo S yva
¨
la
¨
for a critical review of the article. T his work has
been supported by Medical Research Foundation of Tampere
University Hospital, Biomed 2 project PL 963433 and Cancer
Foundation in Pirkanmaa.
References

1. Hsu, S.C. & DeFranco, D.B. (1995) Selectivity of cell cycle reg-
ulation of glucocorticoid r eceptor function. J. Biol. Chem. 270,
3359–3364.
2. Lim-Tio, S.S., Keightley, M.C. & Fuller, P.J. (1997) Determinants
of specificity of transactivation by the mineralocorticoid or glu-
cocortic oid receptor. Endocrinology 138, 2537–2543.
3. Kino, T. & Chrousos, G.P. (2001) Glucocorticoid and miner-
alocorticoid resistance/hypersensitivity syndromes. J. Endocrinol.
169, 437–445.
4. Kurihara, I., Shibata, H., Suzuki, T., Ando, T., Kobayashi, S.,
Hayashi, M., Saito, I. & Saruta, T. (2002) Expression and reg-
ulation of n uc lear re cepto r coac tivators in glucocorticoid action.
Mol. Cell. Endocrinol. 189, 181–189.
5. Leo, C. & C hen, J.D. (2000) The SRC family of nuclear receptor
coactivators. Gene 245, 1–11.
6. Voegel, J.J., Heine, M.J., Zechel, C., Chambon, P . & Gron emeyer,
H. (1996) TIF2, a 16 0 kDa transcriptional m ediator for the lig and-
dependent activation function AF-2 of nuclear receptors. EMBO
J. 15, 3667–3675.
7. Hong, H., Kohli, K., Trivedi, A ., Johnson, D.L. & Stallcup,
M.R. (1996) GRIP1, a novel mouse protein that serves as a
transcriptional coactivator in yeast for the hormone binding
domains of steroid receptors. Proc. N atl Acad. Sci. USA 93, 4948–
4952.
8. Chen, S.L., Dowhan, D .H., Hosking, B.M. & M uscat, G.E. (2000)
The steroid recepto r coactivator, GRIP-1, is necessary for MEF-
2C-dependent gene expression and skeletal muscle differentiatio n.
Genes Dev. 14, 1209–1228.
9. Cavarretta, I.T., Mukopadhyay, R., Lonard, D.M., Cowsert,
L.M., Bennett, C .F., O’Malley, B .W. & Smith, C.L. (2002)

4166 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Reduction of coactivator expression by antisense oligodeoxy-
nucleotides inhibits ERalpha transcriptional a ctivity and MCF-7
proliferation. Mol. Endocrinol. 16, 253–270.
10. Borud, B., Hoang, T., Bakke, M., Jacob, A.L., Lund, J. &
Mellgren, G. (2002) The nuclear receptor coactivators p300/CBP/
Cointegrator-associated protein (p/CIP) and transcription inter-
mediary factor 2 (TIF2) differentially regulate PKA-stimulated
transcriptional activity of steroidogenic factor 1. Mol. Endocrinol.
16, 757–773.
11.Misiti,S.,Schomburg,L.,Yen,P.M.&Chin,W.W.(1998)
Expression and hormonal regulatio n of coac tivator and co-
repressor genes. Endocrinology 139, 2493–2500.
12. Schmidt, P., H olsboer, F. & Spengler, D. (2001) Beta (2)-adre-
nergic receptors potentiate glucocorticoid receptor transactivation
via G prote in beta gamm a-subunits and the p hosphoinositide
3-kinase pathway. Mol. Endocrinol. 15, 553–564.
13. Rohrer, D.K. & Kobilka, B.K. (1998) G prote in-coupled
receptors: function al and mechanistic insights through a ltered
gene expression. Physiol. Rev. 78, 35–52.
14. Morris, A.J. & M albon, C.C. (1999) P hysiological re gulation of G
protein-linked signaling. Physiol. Rev. 79, 1373–1430.
15. Ji, T.H., Grossmann, M. & Ji, I. (1998) G protein-coupled
receptors. I. Diversity o f receptor–ligand interactions. J. Biol.
Chem. 273, 17299–17302.
16. Brady, A.E. & Limbird, L.E. (2002) G protein-coupled receptor
interacting proteins: emerging roles in localization and signal
transduction. Cell Signal. 14 , 297–309.
17. Darmoul, D., Marie, J.C., D evaud, H., Gratio, V. & Laburthe, M.
(2001) Initiation of human colon cancer cell proliferation by

trypsin acting at protease-activated recep tor-2. Br.J.Cancer85,
772–779.
18. Montaner,S.,Sodhi,A.,Pece,S.,Mesri,E.A.&Gutkind,J.S.
(2001) The Kaposi’s sarcoma-associated herpesvirus G protein-
coupled receptor promotes endothelial cell survival through
the activation of Akt/protein kinase B. Cancer Res. 61, 2641–
2648.
19. Weber, H., Webb, M.L., Serafino, R., Taylor, D.S., Moreland, S.,
Norman, J. & Molloy, C.J. (1994) Endothelin-1 and angiotensin-
II stimulate delayed mitogenesis in cultured rat aortic smooth
muscle cells: evidence for common signaling mechanisms. Mol.
Endocrinol. 8, 148–158.
20. Gazvani,R.,Smith,L.&Fowler,P.A.(2002)Effectofinterleukin-
8 (IL-8), anti-IL-8, and IL-12 on en dometrial cell su rvival in
combined endometrial gland and stromal cell cultures derived
from women with and without endometriosis. Fertil. Steril. 77,
62–67.
21. Xu, Y., Zhu, K., Hong, G., Wu, W., Baudhuin, L.M., Xiao, Y. &
Damron, D.S. (2000) Sphingosylphosphorylcholin e is a ligand for
ovarian cancer G-protein-coupled receptor 1. Nat. Cell Biol. 2,
261–267.
22. Xoriuchi,M.,Hamai,M.,Cui,T.X.,Iwai,M.&Minokoshi,Y.
(1999) Cross talk between angiotensin II type 1 and type 2
receptors: cellular mechanism of angiotensin type 2 receptor-
mediated cell growth inhibition. Hyp ertens. Res. 22 , 67–74.
23. Ferjoux,G.,Bousquet,C.,Cordelier,P.,Benali,N.,Lopez,F.,
Rochaix, P., B uscail, L. & Susini, C. (2000) Signal transduction
of somatostatin receptors negatively controlling cell proliferation.
J. Physiol. Paris 94, 205–210.
24. Carmeci, C., T ho mpson, D.A., Ring, H .Z. , F rancke, U. & Weigel,

R.J. (1997) Identification o f a gene (GPR30) w ith h omology to the
G-protein-coupled receptor superfamily associated with estrogen
receptor expression in breast canc er. Genomics 45, 607–617.
25. Takada, Y., Kato, C., Kondo, S., Korenaga, R. & Ando, J. (1997)
Cloning of cDNAs encoding G protein-coupled receptor
expressed in human endothelial cells exposed to fluid shear stress.
Biochem. Biophys. Res. Commun. 240, 737–741.
26. Kvingedal, A.M. & Smeland, E.B. (1997 ) A nove l putative
G-protein-coupled receptor expressed in lung, heart a nd l ymphoid
tissue. FEBS Lett. 407, 59–62.
27. Feng, Y. & Gregor, P. (1997) Cloning of a novel member of the
G protein-coupled receptor family related to peptide rece ptors.
Biochem. Biophys. Res. Commun. 231, 651–654.
28. Owman, C., Blay, P., Nilsson, C. & Lolait, S.J. (1996) Cloning of
human cDNA enc oding a no vel heptahelix receptor e xpressed in
Burkitt’s lymphoma a nd widely distribu ted in b rain and p e ripheral
tissues. Biochem. Biophys. Res. Commun. 228, 285–292.
29. Ahola, T.M., Purmonen, S., Pennanen, P., Zhuang, Y.H.,
Tuohimaa, P. & Ylikomi, T. (2002) Progestin u pregulates
G-protein-coup led receptor 30 in breast c ancer cells. Eur. J. Bio-
chem. 269, 2485–2490.
30. Ahola, T.M., Manninen, T., A lkio, N. & Ylikomi, T. (2002) G
protein-coupled receptor 30 is critical for a progestin-induced
growth inhibition in MCF-7 breast cancer cells. Endocrinology
143, 3376–3384.
31. Kimura, M., Mizukami, Y., Miur a, T., F ujimoto, K .,
Kobayashi, S. & Matsuzaki, M . (2001) Orphan G protein-
coupled receptor, GPR41, induces apoptosis via a p53/Bax p ath-
way during ischemic hypoxia and reoxygenation. J. Biol. Chem.
276, 26453–26460.

32. Filardo, E.J., Quinn, J.A., Bland, K.I. & Frackelton, A.R. Jr
(2000) E strogen-induced activation of Erk-1 and Erk-2 requires
the G protein- coupled receptor homolog, GPR30, and occurs via
trans-activation of the epidermal growth factor recept or through
release o f HB-EGF. Mol. Endocrinol. 14, 1649–1660.
33. Filardo, E.J., Quinn, J.A., Frackelton, A.R. Jr & Bland, K.I.
(2002) Estrogen action via the G protein-coupled receptor,
GPR30. Stimulation of adenylyl cyclase and cAMP-mediated
Attenuation of e pidermal growth factor receptor-to-MAPK sig-
naling axis. Mol. Endocrinol. 16, 70–84.
34. Kueng, W., Silber, E. & Eppenberger, U. (1989) Quantification of
cells cultured o n 96-well plates. Anal. Biochem. 182, 16–19.
35. Ahonen, M.H., Zhuang, Y .H., Aine, R., Ylikomi, T. & Tuohi-
maa, P. (2000) Androgen receptor and vitamin D receptor in
human ovarian cancer: growth stimulation and inhibition by
ligands. Int. J. Cancer 86, 4 0–46.
36. Su, L.F., Wang, Z. & Garabedian, M.J. (2002) Regulation of
GRIP1 and CPB coactivator activity by Rho GDI modulates
estrogen receptor transcriptional enhancement. J. Biol. Chem. 277,
37037–37044.
37. Ahola, T.M., Alkio, N., Manninen, T. & Ylikomi, T. (2002)
Progestin and g protein-coupled receptor 30 inhibit mitogen-
activated protein kinase activity in MCF-7 breast cancer cells.
Endocrinology 14 3 , 4620–4626.
38. Bodwell, J.E., Webster, J.C., Jewell, C.M., Cidlowski, J.A., Hu,
J.M. & Munc k, A. ( 1998) Gluco corticoid recepto r pho sphoryla-
tion: overview, function and c ell cycle-dependence. J. Steroid.
Biochem. Mol. Biol. 65, 91–99.
39. LeDrean,Y.,Mincheneau,N.,LeGoff,P.&Michel,D.(2002)
Potentiation of glucocorticoid receptor transcriptional activity by

sumoylation. Endocrinology 143 , 3482–3489.
40. Hammer, G.D., Krylova, I., Zhang, Y., Darimont, B.D., Simp-
son, K., Weigel, N .L. & I ngraham, H.A. (1999) Phosphorylation
of the nuclear rec eptor SF-1 modulates cofactor rec ruitment:
integration of hormone signaling in reproduction and stress. Mol.
Cell 3, 521–526.
41. Lange, C.A., Shen, T. & Horwitz, K.B. (2000) Phosphorylat ion o f
human progesterone receptors at serine-294 by mitogen-activated
protein kinase signals th eir degradation by the 26S proteasome.
Proc.NatlAcad.Sci.USA97 , 1032–1037.
42. Shen, T., Horwitz, K.B. & Lange, C.A. (2001) Transcriptional
hyperactivity of human progesterone receptors is coupled to their
ligand-dependent dow n-regulation by m itogen-activated p rotein
Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4167
kinase-dependent phosphorylation o f s e rine 294. Mol. Cell. Biol.
21, 6122–6131.
43. Chen, J., Bander, J.A., Santore, T.A., Chen, Y., Ram, P.T., Smit,
M.J. & Iyengar, R. (1998) Expression of Q227L-galphas in MCF-
7 human breast cancer cells inhibits tumorigenesis. Proc. Natl
Acad. Sci. USA 95, 2648–2652.
44. Santore, T.A., Chen, Y., Smit, M.J. & I yengar, R. (2002)
Adenovirus-directed e xpression of Q227L-G alpha (s) inhibits
growth of established tumo rs o f later-stage human breast cancer
cells in athymic mice. Proc. Natl Acad. Sci. USA 99, 1671–
1676.
45.Leo,J.C.,Guo,C.,Woon,C.T.,Aw,S.E.&Lin,V.C.
(2004) Glu cocorticoid and mineralocorticoid cross-talk with
progesterone rec eptor to induce focal a dhesion a nd growth
inhibition in breast cancer cells. Endocrinology, 145, 1314–1321.
46. Hamza, A., S arma, M .H. & Sarma, R.H. (2003) Plausible inter-

action of an alpha-fetop rotein cyclopeptide with the G-protein-
coupled receptor model GPR30: docking study by molecular
dynamics simulated annealing. J. Biomol. Struct. Dyn. 20, 751–
758.
47. Vakharia, D. & Mizejewski, G.J. (2 000) H uman alpha-fetoprotein
peptides bind estrogen receptor and estradiol and suppress breast
cancer. Breast Cancer Res. Treat. 63, 41–52
48. Tilghman, S.M. & Belayew, A. (19 82) Transcriptional control of
the murine albumin /alpha-fetoprot ein locus du ring development.
Proc.NatlAcad.Sci.USA, 79, 5254–5257.
4168 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004