MINIREVIEW
Gonadotropin-releasing hormone: GnRH receptor signaling
in extrapituitary tissues
Lydia W. T. Cheung and Alice S. T. Wong
School of Biological Sciences, University of Hong Kong, China
Introduction
The hypothalamic gonadotropin-releasing hormone
(GnRH) is a decapeptide that plays a critical role in
the regulation of reproduction. GnRH-I (pGlu-His-
Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH
2
) is the first
GnRH isoform discovered in mammalian brain. Its
major role is to stimulate pituitary secretion of
gonadotropins, luteinizing hormone and follicle-stimu-
lating hormone, which in turn stimulate the gonads
for steroid production. Subsequently, a second iso-
form of GnRH (His5, Trp7, Tyr8) (GnRH-II) has
been isolated from chicken brain. It is also highly
conserved among vertebrates, including mammals [1].
However, in contrast to GnRH-I, GnRH-II is
expressed at significantly higher levels outside the
Keywords
cross-talk; extrapituitary; GnRH; GnRH
receptor; MAPK; metastasis; pituitary;
receptor tyrosine kinase; signaling; tumor
Correspondence
A. S. T. Wong, School of Biological
Sciences, University of Hong Kong, 4S-14
Kadoorie Biological Sciences Building,
Pokfulam Road, Hong Kong, China

receptor(s) for GnRH exists.
Abbreviations
EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-related kinase; FAK, focal adhesion
kinase; FGF, fibroblast growth factor; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor;
JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-jB, nuclear factor kappa B; PI3K,
phosphatidylinositol 3-kinase; PKC, protein kinase C; Pyk2, proline-rich tyrosine kinase 2; RTK, receptor tyrosine kinase; uPA, urokinase-type
plasminogen activator; VEGF, vascular endothelial growth factor.
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5479
brain and is particularly abundant in the kidney,
bone marrow, and prostate [2]. This leads to the
speculation that GnRH-II may have distinct physio-
logical functions from those of GnRH-I. In line with
this is the observation that although GnRH-II can
stimulate gonadotropin secretion, its efficiency is
much lower than that of GnRH-I (only about 2% of
that of GnRH-I) [3]. This suggests that the primary
role of GnRH-II is not in the regulation of gonado-
tropin secretion. Instead, this peptide has been shown
to act as a neuromodulator [4]. The exact actions of
GnRH-II in peripheral tissues are not entirely under-
stood, but this is certainly an important topic for
investigation which may offer an opportunity to eluci-
date the undisclosed complexity of GnRH.
In this minireview, we will focus on recent progress
in understanding the roles of GnRH-I and GnRH-II
in extrapituitary tissues, in particular its emerging
role in tumor growth, invasion, and metastasis. We
will also describe the molecular mechanisms underlying
these effects, focusing on the roles of proteolysis,
adhesion, and signaling, as well as our still-emerging

Interestingly, levels of GnRHR seem to be associated
with cancer grading and have been reported to be
elevated in advanced stage (stages III and IV) as
compared to early stage (stages I and II) ovarian
carcinomas [11]. Our recent findings that GnRH can
promote the motility and invasiveness of ovarian can-
cer cells further corroborate the view that GnRH may
play a crucial role in tumor progression ⁄ metastasis
[12,13], and these findings will be discussed in a later
section.
Using [
125
I][d-Trp6]GnRH, specific receptor binding
has been detected in membranes from 24 of 31 (77%)
endometrial carcinomas and from three of 13 (23.1%)
nonmalignant human endometrial specimens [14].
GnRHR mRNA has been clearly detected in surgical
endometrial carcinoma specimens and endometrial
carcinoma cell lines [15,16]. As with normal myome-
trium, most benign neoplasms studied thus far,
including uterine leiomyoma, also possess GnRHR
[17].
Early studies showed that the human placenta con-
tains specific binding sites for GnRH that interact with
GnRH agonists and antagonists [18]. Later on,
GnRHR was localized to the cytotrophoblast and
syncytiotrophoblast cell layers [19,20]. Temporal
expression of GnRHR in the placental cells at different
weeks of gestation has been observed, in parallel with
the time-course of chorionic gonadotropin secretion

GnRHR ligands on the gonadotropes. Second, there
are at least two classes of GnRHR: one has high affin-
ity [with nanomolar dissociation constants (K
d
)] for
GnRH, and one has low affinity (with micromolar K
d
values) for GnRH. The high-affinity GnRH-binding
sites are commonly regarded as being the same as the
GnRHR of the pituitary gland. Whereas in most of
the reported cases, both the low-affinity and high-affin-
ity GnRHR have been found in extrapituitary tissues
[30–33], in some cases, only low-affinity GnRHR could
be detected [10,18,34], and in others, e.g. in endome-
trial cancers and nonmalignant endometrial specimens,
only the high-affinity GnRHR has been demonstrated
[14]. The exact role of each of these receptors and the
implications of differential levels of expression remain
to be elucidated.
Functions of GnRH-I and GnRH-II in
cancers
Tumor growth
Over the last two decades, both GnRH agonists and
antagonists have been widely used as therapeutics in
treating sex steroid-dependent tumors. The majority
of these GnRH analogs, when given continuously,
inhibit gonadotropin synthesis and secretion via
downregulation of the pituitary GnRHRs. This indi-
rect mechanism of action has provided the rationale
for the use of GnRH analogs in the treatment of hor-

the effects of GnRH-I antagonists are stronger than
those of the agonists [44]. This difference has also been
seen in an in vivo model, which demonstrates a signifi-
cant inhibition of tumor growth by GnRH-I antago-
nists but not GnRH-I agonists [45]. The advantage of
GnRH antagonists over the agonistic peptides is prob-
ably due to the fact that they inhibit the secretion of
gonadotropins and reduce sex steroid levels immedi-
ately after application, thus achieving rapid therapeutic
effects, whereas repeated exposure to agonistic agents
is required to induce functional desensitization of the
gonadotropes [46].
Treatment of human endometrial cancer cells (cell
line Ishikawa) with the GnRH-I antagonist SB-75
results in growth inhibition, mainly due to the Fas ⁄ Fas
ligand-mediated apoptotic pathway, whereas GnRH-I
agonists have no effect on the same cell line [15,47,48].
Another endometrial carcinoma cell line, HEC-1A, also
exhibits differential responses to different GnRH agon-
ists and antagonists [15,30,36,48]. GnRH-II has been
shown to have antiproliferative effects on endometrial
carcinoma cells [41]. The effects of GnRH-I are
abrogated after type I GnRHR knockout [36], whereas
those of the GnRH-I antagonist cetrorelix and of
GnRH-II persist [41]. These findings suggest that the
antiproliferative effects of cetrorelix and GnRH-II are
not mediated through the type I GnRHR.
GnRH-I has been demonstrated to have antiprolifer-
ative effects on prostate cancer cells [49–51], except in
one in vivo study [52]. This antiproliferative effect

trophoblasts and decidual stromal cells to facilitate
implantation [59,60]. However, its potential role in
cancer metastasis has just begun to be revealed.
Metastasis is a complex phenomenon that requires
several specific steps, such as decreased adhesion,
increased motility, and proteolysis. The effects of GnRH
in tumor metastasis are mediated through the regulation
of adhesion molecules, Rho GTPases, and two families
of metastasis-related proteinases, the matrix metallopro-
teinases (MMPs) and the urokinase-type plasminogen
activator (uPA) system, at several levels: mRNA
transcription, secretion, and proenzyme activation.
The ability of GnRH to regulate metastasis was first
reported in melanoma cells [61]. High doses of GnRH-I
analog, at micromolar concentrations, significantly
reduces the ability of melanoma cells to invade
and migrate [61]. Preliminary data (R. M. Moretti,
M. Monagnani Marelli, J. C. van Groeninghen, M.
Motta & P. Limonta, unpublished results, 2003) indicate
that this inhibitory action is due to the effects of
integrins and MMPs [62].
We were the first to report possible metastatic activ-
ity of GnRH-I in tumors of the female reproductive
tract [12]. GnRH-I exerts a biphasic effect on cellular
migration and invasion: whereas lower (nanomolar)
concentrations of the GnRH-I agonist stimulate cellu-
lar migration and invasion in a dose-dependent man-
ner, high (micromolar) concentrations are not as
efficient. This proinvasive effect is mediated through
activation of metastasis-related proteinases, in particu-

cancer metastasis, using a coculture system with
human osteosarcoma cells to analyze tumor cell
invasion to bone [68]. The consequences of GnRHR
activation are complex and appear to be cell context
dependent: whereas treatment of cells with the
GnRH-I agonist triptorelin, the GnRH-II agonist
[d-Lys6]GnRH-II and the GnRH-I antagonist cetrorelix
decreases the invasion rate in most breast cancer cell
lines, these agents have no significant effect in the
GnRHR-positive MDA-MB-435 cells [68]. Further
investigations are required to elucidate the reason why
the MDA-MB-435 cell line reacts differently.
Organ-specific homing and colonization of cancer
cells are important and interesting features of metasta-
sis. A role for GnRH has also been suggested in the
regulation of the immune response and metastasis.
GnRH-I and GnRH-II are expressed in human normal
and cancerous T-cells. GnRH triggers laminin receptor
gene expression, adhesion to laminin, in vitro chemo-
taxis, and in vivo homing to specific organs [69].
Angiogenesis
Angiogenesis is crucial to a number of physiological
and pathological processes, such as reproduction,
development, and tissue repair, as well as tumor
growth and metastasis. Vascular endothelial growth
factor (VEGF) is implicated as the most important
angiogenesis inducer, because of its potency in a
variety of normal and tumor cells. Other angiogenic
factors include fibroblast growth factor (FGF), plate-
let-derived growth factor and the angiopoietin family.

of GnRH analogs. Rather, GnRHRs couple to G
ai
in
these tumors and result in the activation of several
downstream signaling cascades [73,74], such as mito-
gen-activated protein kinase (MAPK), phosphatidyl-
inositol-3-kinase (PI3K), and nuclear factor kappa B
(NF-jB) signaling. The GnRH-induced signaling path-
ways in extrapituitary tissues are shown schematically
in Fig. 1.
Fig. 1. Schematic representation of GnRHR signaling in extrapituitary tissues. Binding of GnRH to GnRHR triggers several intracellular signal-
ing cascades and cross-talk with mitogenic signaling, depending on the cell context. Some of these signaling modules can transduce extra-
cellular signals to the nucleus and thereby regulate genes that are involved in cell growth, metastasis, or survival. Arr, b-arrestin; CREB,
cAMP response element-binding protein; FGFR, fibroblast growth factor receptor; HB-EGF, heparin-binding EGF; IjB, inhibitory factor kap-
pa B; IGFR, IGF receptor; MEK, mitogen-activated protein kinase kinase; MLK3, mixed-lineage kinase 3; PTP, protein tyrosine phosphatase;
Sos, son of sevenless; TNF-a, tumor necrosis factor alpha.
L. W. T. Cheung and A. S. T. Wong GnRH receptor signaling
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5483
MAPK
The major MAPK cascades include extracellular sig-
nal-regulated kinase (ERK), Jun N-terminal kinase
(JNK), and p38 MAPK. Many studies have shown
that the MAPK pathway is critical for GnRH activi-
ties, which provides an important link for the trans-
mission of signals from the cell surface to the nucleus.
Activation of MAPK by GnRH involves distinct
upstream pathways in generating tissue-specific and
cell-specific signaling (Fig. 1). This can occur at differ-
ent levels via different mechanisms: (a) second messen-
gers [protein kinase C (PKC) and cAMP] [26];

ovarian cancer cell line, OVCAR-3. Both ERK1 ⁄ 2 and
p38 MAPK mediate the antiproliferative effects of
GnRH-I and GnRH-II in a PKC-dependent manner
[43,77]. GnRH-II induces the activation of activator
protein-1 transcription factor via p38 MAPK, suggest-
ing a potential role of activator protein-1 in ovarian
cancer cell growth [77]. The JNK pathway also drives
tumor invasion and migration in ovarian cancer cells
[12], but the activation mechanism(s) remains to be
elucidated.
Temporal and spatial differences in cellular signaling
may have significant phenotypic manifestations [78,79].
For example, sustained activation of ERK1 ⁄ 2 has been
implicated in nerve growth factor-mediated neuronal
differentiation of PC12 cells, whereas a rapid and
transient activation is associated with growth factor-
mediated proliferation of PC12 cells [80]. Thus, the
duration of kinase activation seems to be a major
determinant of signal outcome. We have shown differ-
ential regulation of ERK1 ⁄ 2, p38 MAPK, and JNK
by GnRH-I with sustained signaling through the JNK
pathway in ovarian cancer cells [12]. Consistently,
GnRH-stimulated MMP-2 and MMP-9 expression,
secretion and cell invasion were attenuated by specific
inhibition of JNK but not of ERK1 ⁄ 2 and p38
MAPK, suggesting that prolonged activation of JNK
may contribute to a more invasive phenotype. Strong
and sustained activation of MAPK has been reported
to be necessary for its cytoplasm-to-nucleus transloca-
tion, and thereby contributes to the regulation of gene

signaling in many reproductive tumor cells [57,66,85–
87]. In prostate cancer cells, cetrorelix is able to coun-
ter EGFR-dependent adhesive signaling through
a PKC-dependent mechanism [66]. Activation of
GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5484 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
GnRHR appears to mediate these effects via activation
of phosphotyrosine phosphatase, thereby reducing
EGF-induced EGFR autophosphorylation, resulting in
downregulation of mitogenic signal transduction and
cell proliferation [85,86,88]. A negative regulatory
interaction between GnRHR and mitogenic signaling
pathways has also been reported in human prostate
cancer cells via insulin-like growth factor. GnRH-I
agonists inhibit expression of the insulin-like growth
factor receptor, receptor tyrosine phosphorylation, and
the subsequent downstream activation of Akt [89–91].
Another example is FGF-2. GnRH analog treatment
has been shown to block cell proliferation and inva-
sion induced by FGF-2 stimulation [65].
PI3K
The PI3K signaling pathway and its downstream
target Akt (also named protein kinase B) has been
implicated in promoting cell survival, proliferation,
and invasion. In uterine leiomyomas, the GnRH-I ago-
nist leuprolide acetate causes a significant reduction in
PI3K ⁄ Akt activity and inhibits the expression of the
antiapoptotic proteins (c-FLIP and PED ⁄ PEA15),
thereby inducing apoptosis [92]. In the SKOV-3 ovar-
ian cancer cell line, GnRH-I and GnRH-II interfere

tor alpha-induced NF-jB signaling in endometriotic
stromal cells (Fig. 1) [95]. These data suggest that
modulation of cytokine signal transduction by GnRH
may be one of the mechanisms contributing to its
growth-inhibitory effect.
The non-RTKs focal adhesion kinase (FAK) and
proline-rich tyrosine kinase 2 (Pyk2) are the predomi-
nant mediators of integrin signaling. GnRHR has been
shown to signal through these molecules, suggesting a
role for GnRH in cytoskeletal reorganization. In
human endometrial cancer cells (HEC-1A), b
3
-integrin-
dependent activation of FAK is associated with the
inhibitory effects of GnRH-I and GnRH-II on growth
[96]. Leiomyoma regression induced by GnRH-I agon-
ists has been suggested to be mediated, at least in part,
through a mechanism involving suppression of FAK
[97]. Maudsley et al. demonstrated a novel signaling
cascade of GnRHR that functionally antagonizes the
actions of testosterone and inhibits prostate tumor
growth [98]. GnRH controls the tyrosine phosphoryla-
tion status of the focal adhesion proteins Pyk2 and
Hic-5. This alteration of the focal adhesion dynamics
then results in nuclear translocation of the androgen
receptor, which renders it transcriptionally inactive
[98].
Mechanisms underlying the diverse
responses to GnRH action
As discussed earlier, GnRH and its agonists have a

mone to stimulate pituitary gonadotropes. On the
other hand, sustained administration of the peptide
brings about a short initial stimulation that is rapidly
followed by a decrease in gonadotropin synthesis and
secretion [99]. In support of this, the signal response is
different at different doses. It has been shown that pul-
satile GnRH stimulates more sustained ERK activity
(more than 8 h), whereas continuous infusion of aT3-1
cells with GnRH stimulates short-term (2 h) ERK
activity [100]. There is also evidence that GnRH treat-
ment stimulates cAMP production at nanomolar con-
centrations, but has an inhibitory effect at micromolar
concentrations [101]. It should be pointed out that the
nanomolar concentration (0.01–1 nm) corresponds to
the physiological circulating level, and the effects
caused by this dose range may represent the physiolog-
ical functions of GnRH [54,102].
Second, GnRH action has been shown to be medi-
ated by coupling to different G
a
-proteins, depending
on the time and dose of exposure [101,103]. In general,
G
aq
and G
as
are associated with a stimulatory effect
[103], whereas G
ai
often mediates the antiproliferative

whereas the a-subunit of G
i
inhibits adenylyl cyclase
activity, the bc-subunits may stimulate the activities of
some adenylyl cyclase subtypes [108,109].
The receptor expression level is also known to be a
determinant for different signal outcomes [6,110,111].
In gonadotropes, different cell surface densities of
GnRHR result in the differential regulation of luteiniz-
ing hormone and follicle-stimulating hormone subunit
gene expression by GnRH-I [112]. We and others have
previously shown that low doses of GnRH upregulate
the expression of its receptor, whereas high doses
decrease it [12,111,113]. This difference in regulation
suggests that high levels of GnRHR expression may
enhance the cellular response to GnRH stimulation,
presumably due to more efficient signal amplification
or altered signaling through coupling to different
G-proteins.
Moreover, ligand selectivity has been proposed to
explain the opposite (stimulatory and inhibitory)
effects of GnRH. For instance, in positively respond-
ing prostate carcinoma cell lines, GnRH-I is more
effective than GnRH-II. On the other hand, in nega-
tively responding cell lines, GnRH-II is much more
effective than GnRH-I. Given the short plasma half-
life of GnRH, efforts have been made to obtain
GnRH analogs, to resist degradation and to increase
potency. However, the different GnRH agonists may
selectively stabilize different receptor-active conforma-

GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5486 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
Table 2. Physiological characteristics and responses to GnRH of common cancer cell lines. fl, decrease; ›, increase; –, no effect; ?, undetermined.
Cell line Origin Isolation
Original
histology
Tumorigenicity
in nude mice
Anchorage-
independent
growth
Invasive
capability
in vitro
Effect of
GnRH on
growth
Effect of GnRH
on motility ⁄
invasion References
CaOV-3 Ovary Primary
carcinoma
Serous Yes High Low fl› [12,76,125,126]
DU-145 Prostate Brain Poorly
differentiated
Yes Low High fl;– fl [50,55,67,120,127,128,132]
HEC-1A Endometrium Primary
carcinoma
Moderately
differentiated

L. W. T. Cheung and A. S. T. Wong GnRH receptor signaling
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5487
amounts of the cotransfected splice variant cDNA and
possibly by preventing or diverting the normal process-
ing of GnRHR or enhancing GnRHR degradation
[118].
Finally, it is also possible that differences in
response may be ascribed to the intrinsic properties of
the cells. The physiological characteristics of the
human cancer cell lines mentioned in this minireview
are summarized in Table 2. For example, in contrast
to SKOV-3 cells, OVCAR-3 cells have low invasive
potential. Thus, whereas low doses of GnRH-I and
GnRH-II can exert a significant invasive effect in
OVCAR-3 cells, they fail to stimulate SKOV-3 maxi-
mally [12,13]. Both GnRH-I and GnRH-II only exert
inhibitory effects on SKOV-3 cells at high doses [13].
Novel receptor(s) for GnRH in humans?
An important issue that remains unresolved in this
field is whether one or more other GnRHR subtypes
exist in humans. The discovery of GnRH-II has stimu-
lated the search for a cognate type II GnRHR. Molec-
ular cloning of the type II GnRHR in goldfish,
marmoset and monkey has shown that the type II
receptor is structurally and functionally distinct from
the type I receptor [138–140]. In humans, a type II
GnRHR has not been found. However, a search of the
human genome database has revealed a putative
type II GnRHR gene on chromosome 1q21.1
[140,141]. Expression of this type II GnRHR mRNA

GnRH-II on mammalian behavior is not mediated via
the type I receptor in musk shrews [147]. Thus, it
appears that GnRH-II may selectively interact with
different GnRHRs to mediate its different actions, pre-
sumably due to the structural differences between the
two GnRHR subtypes.
Alternatively, it is possible that the human type II
GnRHR may be encoded by a different gene that has
yet to be identified. Database searches have revealed
the presence of more than two other GnRHR genes in
the human genome apart from the conventional type I
receptor gene [148]. These genes are located on sepa-
rate chromosomes. Whether functional, full-length
transcripts can be produced from these receptor-like
genes remains to be determined. Recently, a novel
GnRH-II-binding protein, in addition to a conven-
tional GnRHR, has been identified by using photo-
affinity labeling with an azidobenzoyl-conjugated
GnRH-II in prostate cancer cells [149]. Taken together,
these observations thus suggest the potential existence
of novel receptors for GnRH-I and GnRH-II.
Concluding remarks
This overview shows that GnRH modulates a variety
of cellular functions in extrapituitary tissues, such as
cell growth, invasion, and angiogenesis. However, the
effects of GnRH are complex and appear to be cell
context dependent. The ability of GnRH to elicit very
different, even opposite (positive and negative),
responses in extrapituitary tissues may arise from dif-
ferential usage of signal transduction pathways and

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