Journal of Neuroendocrinology, 2012, 24, 1463–1475

© 2012 The Authors.

REVIEW ARTICLE Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology

Gonadotrophin-Releasing Hormone Signalling Downstream of Calmodulin

P. Melamed*†, D. Savulescu*, S. Lim†, A. Wijeweera*, Z. Luo†1, M. Luo†2 and L. Pnueli*
*Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
†Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

Journal of Gonadotrophin-releasing hormone (GnRH) regulates reproduction via binding a G-protein

Neuroendocrinology
Correspondence to:
P. Melamed, Faculty of Biology,
Technion-Israel Institute of
Technology, Haifa 32000, Israel
(e-mail: philippa.melamed@gmail.
com).
1
Present address: Stowers Institute for
Medical Research, Kansas City MO,
USA.
2
Present address: Center for Cell and
Gene Therapy, Baylor College of
Medicine, Houston TX, USA
coupled receptor on the surface of the gonadotroph, through which it transmits signals, mostly
via the mitogen-activated protein (MAPK) cascade, to increase synthesis of the gonadotrophin
hormones: luteinising hormone (LH) and follicle-stimulating hormone (FSH). Activation of the
MAPK cascade requires an elevation in cytosolic Ca2+ levels, which is a result of both calcium
influx and mobilisation from intracellular stores. However, Ca2+ also transmits signals via an
MAPK-independent pathway, through binding calmodulin (CaM), which is then able to bind a
number of proteins to impart diverse downstream effects. Although the ability of GnRH to
activate CaM was recognised over 20 years ago, only recently have some of the downstream
effects been elucidated. GnRH was shown to activate the CaM-dependent phosphatase,
calcineurin, which targets gonadotrophin gene expression both directly and indirectly via
transcription factors such as nuclear factor of activated T-cells and Nur77, the Transducer of
Regulated CREB (TORC) co-activators and also the prolyl isomerase, Pin1. Gonadotrophin gene
expression is also regulated by GnRH-induced CaM-dependent kinases (CaMKs); CaMKI is able to
derepress the histone deacetylase-inhibition of b-subunit gene expression, whereas CaMKII
appears to be essential for the GnRH-activation of all three subunit genes. Asides from
activating gonadotrophin gene expression, GnRH also exerts additional effects on gonadotroph
function, some of which clearly occur via CaM, including the proliferation of immature gonado-
trophs, which is dependent on calcineurin. In this review, we summarise these pathways, and
discuss the additional functions that have been proposed for CaM with respect to modifying
GnRH-induced signalling pathways via the regulation of the small GTP-binding protein, Gem,
and/or the regulator of G-protein signalling protein 2.
Key words: GnRH, gonadotrophin, gonadotroph, calmodulin, calcineurin, CaMK
doi: 10.1111/j.1365-2826.2012.02359.x

these cells in numerous other ways, including cell proliferation and

Gonadotrophin-releasing hormone (GnRH), the gonadotroph
and calmodulin
Reproduction is regulated through a complex interplay of hormones
and feedback operating along the hypothalamic-pituitary-gonadal
axis, of which the hypothalamic hormone, gonadotrophin-releasing
hormone (GnRH), is the predominant activator. GnRH binds a
membrane-bound receptor (GnRHR) on the pituitary gonadotrophs
to stimulate transcription of the three gonadotrophin genes: the
common a subunit (aGSU) and the hormone specific b-subunits
[luteinising hormone (LH)b and follicle-stimulating hormone (FSH)b]
(Fig. 1), as well as stimulating the secretion of LH. As part of its
regulation of gonadotroph activity and function, GnRH also affects
apoptosis, cell shape and mobility, as well as their responsiveness
to other hormones (1–6).
The GnRH arrives at the pituitary in pulses whose amplitude and
frequency vary with ensuing sexual maturity and during the
oestrous cycle; these distinct pulse frequencies direct the preferen-
tial expression of either LHb or FSHb (7–10). Subsequent to their
synthesis, the gonadotrophic hormones, LH and FSH, are secreted
into the circulation from where they regulate gonadal activity. The
pivotal role of GnRH in functioning of the gonadotrophs and the
regulation of reproduction is seen at puberty, when GnRH delivery
to the gonadotroph is the only endogenous block to the reawaken-
ing of the pituitary-gonadal axis and reproductive potential (11).
1464 P. Melamed et al.

GnRH
Ca2+
GnRHR
as aq
PKA
Ca2+
Ca2+ PLCb
Calmodulin

PKC
CaMKI CaMKII Calcineurin
P
ERK1/2
Pin1
14-3-3 P P
P TORC NFAT3
HDAC4
Cytosol

Nucleus
HDACsHDAC4 CoR Pin1 SF-1

Gonadotrophin b-subunit gene TORC CDK7

transcription (LHb and FSHb)
NFAT4 SF-1 NFAT3 AP-1 P
SF-1

U
–P TORC U
Gonadotrophin gene
transcription (GSU)
P U P P
Nur77 CREB AP-1 Pitx1 SF-1 Egr-1

Gonadotrophin gene transcription (LHb )

Gonadotrophin gene transcription (FSHb )

Fig. 1. Model of gonadotrophin-releasing hormone (GnRH)-induced pathways downstream of calmodulin towards gonadotrophin gene expression. Upon bind-
ing its G-protein coupled receptor on the cell membrane, GnRH induces calcium influx (as well as mobilisation: not shown), which activates calmodulin (CaM).
Some of the effects downstream of CaM via the CaM kinases and the phosphatase, calcineurin, which lead to gonadotrophin gene expression, are shown. Solid
lines represent effects that are likely direct, and dashed lines represent pathways that may or may not have intermediate elements that are not shown. Blue
lines represent transcriptional, and black lines post-translational effects on the target. The pink double-headed arrows indicate proteins that shuttle between
cellular compartments. CaMK, calmodulin-dependent kinase; CoR, receptor corepressor; ERK, extracellular-signal regulated kinase; HDAC, histone deacetylase;
NFAT, nuclear factor of activated T-cells; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; TORC, transducer of regulated CREB.

Upon binding to its G-protein coupled receptor on the gonado-
troph, GnRH induces the production of inositol triphosphate (IP3)
and diacylglycerol for downstream signalling, much of which is via
protein kinase C (PKC) (12). The elevation of IP3 results in calcium
mobilisation from the endoplasmic reticulum (ER), with external
calcium also entering through voltage-gated calcium channels on
the cell membrane. These GnRH-induced events trigger the major
mitogen-activated protein kinase (MAPK) cascades, culminating in
activation of extracellular-signal regulated kinase (ERK) 1/2, c-Jun
NH2-terminal kinase (JNK), p38 MAPK and ERK5 (10,12,13). The ele-
vation in available intracellular Ca2+ is temporary because the Ca2+
is quickly sequestered by binding proteins and is also pumped out
of the cytosol after inhibition of the ER IP3-sensitive Ca2+ channels
by the high levels of cytosolic Ca2+ (14). The resulting short pulses
of free Ca2+ play crucial roles in cell signalling in many cell types,
and their roles in activating regulated LH secretion have been docu-
mented extensively (14,15), while also playing additional roles in
the regulation of other aspects of cell function and gene expression
(16,17). Diverse studies have indicated the varying involvement of
this Ca2+ in expression of the gonadotrophin genes (12) and it was
suggested that Ca2+ sensing might play a role in the decoding of
GnRH pulse-frequency (18).
Apart from its role in the activation of PKC and the downstream
MAPK pathways, Ca2+ is also able to induce distinct cellular out-
comes via additional binding protein effectors, most notably cal-
modulin (CaM), which is expressed ubiquitously. CaM does not
contain its own enzymatic activity but, in different cell contexts,
interacts with a variety of proteins, including kinases, phosphatases
and ion channels to transduce the signalling effects to diverse
pathways (19). This is possible because the CaM N-terminal and
C-terminal domains are connected by a linker region which allows
it to assume distinct conformations when bound to different
targets. In the absence of Ca2+, it adopts a closed conformation
but, after Ca2+ binding, the conformational change exposes more
hydrophobic groups, thus enhancing the ability of CaM to interact
with the various effector proteins (19-21).
Early studies in gonadotrophs demonstrated that GnRH induces
a change in localisation of CaM from the cytoplasm to the cell
membrane and also indicated that it has a role in GnRH-induced
LH release, as well as in the expression of primary response genes,

© 2012 The Authors. Journal of Neuroendocrinology, 2012, 24, 1463–1475

Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology

c-fos, c-jun and junB, (22–24). More recently, two of the better
characterised signalling pathways downstream of CaM, the CaM-
dependent kinases (CaMKs) and the phosphatase, calcineurin, have
been shown to play roles in transducing GnRH-activated signals to
gonadotrophin gene transcription and other aspects of gonadotroph
function. In this review, we summarise these pathways, and
examine other evidence for a role of CaM in modulating GnRH-
induced ERK signalling, possibly involving the regulator of G-protein
signalling (RGS) protein, RGS2, or the small GTP-binding protein,
Gem.
CaMKs and gonadotrophin gene expression
The CaMKs are serine/threonine kinases, which include CaMKI, CaM-
KII and CaMKIV, as well as the upstream CaMK kinase (CaMKK).
These kinases have comprehensive functions and are involved in
gene expression, cell proliferation, apoptosis and cytoskeletal reor-
ganisation, as well as synaptic development and plasticity and
behaviour (21,25). CaMKI, CaMKII and CaMKK are widely expressed,
whereas CaMKIV is more tissue-restricted and is not found in the
gonadotrophs (26).
All of the CaMKs contain a Ca2+/CaM -binding region that over-
laps with an autoinhibitory domain. Binding of Ca2+/CaM disrupts
the interaction between the autoinhibitory domain and the catalytic
domain, thus activating the kinase (21). CaMKK is an upstream acti-
vator of CaMKI and CaMKIV that shares similar structure with the
other CaMKs; however, after its initial activation, the CaMKKb iso-
form can also function independently of Ca2+/CaM because of the
release of its autoinhibitory domain; moreover, it may also have
some autonomous activity through its autophosphorylation (21,27).
This isoform also acts as a scaffold for a signalling complex includ-
ing CaM and the CaMKK substrate, which was recently shown to
include AMP-dependent kinase (AMPK) (28). Interestingly, GnRH
was shown to induce a prolonged increase in AMPK activity in the
gonadotrophs (29), although a role for CaMKK in this activation
has yet to be reported.
CaMKI
The activity of CaMKI is entirely dependent on activation by Ca2+/
CaM, and CaMKI activity drops as soon as the levels of Ca2+ return
to basal levels. This dependency is a result of fact that the Ca2+/
CaM-induced displacement of the autoinhibitory domain is required
to expose the threonine that is targeted by CaMKK. Phosphorylation
of this site by CaMKK increases CaMKI activity, probably by enhanc-
ing the stability of the activation loop that increases the compe-
tency of the catalytic site (21).
CaMKI is expressed in the aT3-1 partially-differentiated immorta-
lised gonadotroph cells and is notably phosphorylated within 5 min
of GnRH treatment; this elevated state is still seen after 60 min of
continuous GnRH exposure (26). CaMKI has a diverse set of protein
targets, including transcription factors and the class 2a histone
deacetylases (HDACs), which shuttle between the nucleus and
cytoplasm. The CaMK-mediated phosphorylation of these class 2a
HDACs was shown to signal binding of the HDAC by 14-3-3
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Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology
GnRH signalling downstream of calmodulin 1465
chaperone proteins that mask the nuclear localisation signal on the
HDAC and may expose a nuclear export signal before exporting the
HDAC out of the nucleus (30–32). HDAC4 and HDAC5 are phos-
phorylated after GnRH exposure and this is almost completely abol-
ished by a dominant negative (dn) CaMKI. Moreover, over-
expression of the dnCaMKI also abolished the ability of GnRH to
de-repress expression of the FSHb gene, although not the LHb gene
(26,33). GnRH causes dissociation of these HDACs from the b-sub-
unit gene promoters, and their nuclear export is dependent on the
serines targeted by CaMKI (Fig. 1). Given that knockdown of HDAC4
alone was sufficient to allow FSHb gene expression, it appears that
GnRH-activated CaMKI mediates the de-repression of this gene in
immature gonadotrophs via targeting these HDACs to cause their
removal from the gene promoter. Interestingly, however, this effect
appears to be unique to the FSHb gene because CaMK inhibition
failed to block the effect of GnRH on the LHb gene, indicating that
GnRH likely directs de-repression of the LHb gene downstream of
CaMK activation (26,33).
CaMKII
CaMKII differs in structure from the other CaMKs and, unlike other
CaMKs, it does not act as a monomer but forms part of a complex
of 12 or more subunits with at least six catalytic domains extend-
ing away from the core. The ability of this complex to contain
diverse proteins likely endows its ability to activate multiple down-
stream effects (21). Similar to other CaMKs, CaMKII also contains
an autoinhibitory domain that blocks catalytic activity until Ca2+/
CaM levels are increased. After the initial activation of one subunit
of the holoenzyme by Ca2+/CaM, it can then phosphorylate a
neighbouring Ca2+/CaM-bound subunit in the same complex. This
phosphorylation disrupts the interaction between the autoinhibitory
domain and the catalytic site, such that the subunit subsequently
retains activity independent of Ca2+/CaM-binding, at the same time
as also increasing the affinity of CaMKII for Ca2+/CaM. Thus, Ca2+/
CaM must bind two molecules in the same holoenzyme to attain
initial activation of CaMKII, which reflects the amount of available
Ca2+; however, subsequent CaMKII activity is maintained even after
transiently-elevated Ca2+ levels drop (21,34). Because of the unique
characteristics of CaMKII, which render it highly sensitive to Ca2+
pulse frequency, it was proposed that CaMKII might provide a
mechanism of decoding Ca2+ oscillations that occur in response to
diverse stimuli (35,36).
GnRH activates CaMKII in rat primary pituitary cells and in go-
nadotroph cell lines, and treatment with the CaMK inhibitor, KN-93,
decreased the GnRH induction of promoter activity and/or mRNA
levels of all three gonadotrophin subunit genes (9,26,37,38). More-
over, the differential expression of these genes was mimicked by
the administration of Ca2+ pulses at frequencies similar to those
seen after GnRH treatment (18). It was therefore suggested by sev-
eral groups that Ca2+/CaM- CaMKII might play a role in decoding
GnRH pulse frequency towards gonadotrophin gene expression. In
one model, it was proposed that CaMKII activation might favour
aGSU and LHb gene expression, which are dominant at higher
GnRH pulse frequencies, whereas at lower frequencies that favour
© 2012 The Authors.
1466 P. Melamed et al.
FSHb transcription, the activated CaMKII, which might be less cru-
cial for FSHb expression, would have time to decay (8). However, a
latter study showed that CaMKII activation by GnRH at 30- or
240 min-pulses, which are optimal for LHb or FSHb activation,
respectively, is not frequency-dependent (9). Moreover, a number of
subsequent studies, both experimental and computational, have
indicated that feedback may be more crucial to the frequency
decoding, and have implicated MAPK phosphatases to play a key
role (10,39,40), whereas feedback inhibition of gene-specific tran-
scription factors has also been suggested (41,42).
The ability of the CaMKII inhibitor to reduce GnRH-induced tran-
scription of all three gonadotrophin b-subunit genes indicates that,
even if it does not decode the GnRH pulse frequency, CaMKII does
mediate some of the effects of GnRH, although its targets in this
pathway largely remain to be elucidated. Recently, however, GnRH-
activation of c-fos was shown to occur via CaMKII phosphorylation
of serum response factor, which likely plays a role in GnRH induc-
tion of FSHb and possibly aGSU transcription (43–46).
Diverse roles for calcineurin in the gonadotroph
Calcineurin is a Ca2+/CaM-dependent serine/threonine protein phos-
phatase comprising two subunits: the catalytic calcineurin A sub-
unit (CnA), and a Ca2+-binding regulatory subunit, calcineurin B
(CnB). The CnA contains three regulatory domains: the CaM-binding
domain, the autoinhibitory domain and a domain that interacts
with CnB. In the absence of Ca2+/CaM, the autoinhibitory domain
binds in the active site and inhibits phosphatase activity. A rise in
intracellular Ca2+, which binds CnB, facilitates Ca2+/CaM binding by
the CaM-binding domain of CnA, resulting in a displacement of the
autoinhibitory domain and activation of the phosphatase, which is
subsequently independent of CnB (47,48).
CnA expression levels increase after GnRH treatment in both aT3-
1 cells and the more fully differentiated immortalised gonadotrope
LbT2 cells (26,49,50). The activity of calcineurin, as well as other
phosphatases, in mediating GnRH signals to gonadotrophin genes
are clearly tightly regulated by GnRH and likely crucial for maintain-
ing the integrity of the signal (10,40,51–54). It has been proposed
that a continual phosphorylation/dephosphorylation cycle of key
substrates is required for GnRH stimulation of gonadotrophin
genes to avoid desensitisation, that they act to down-regulate and
constrain the magnitude or duration of GnRH signalling, and also
that they play a role in decoding the GnRH pulse frequency
(10,40,51,55,56). However, unlike the MAPK phosphatases, the effects
of calcineurin in these cells all appear to be stimulatory to gonado-
trophin gene expression and are mediated through multiple
targets. Inhibition of calcineurin is repressive to transcription and/
or promoter activation of all three gonadotrophin subunits
(26,50,54,57). For the aGSU, calcineurin is both necessary and suffi-
cient to induce gene expression, whereas, for FSHb, it is necessary
but not sufficient, although it clearly augments the effect of GnRH.
For the LHb gene, its exact role remains to be elucidated, although it
likely includes indirect effects such as those occurring via Pin1,
which also impacts other aspect of gonadotroph cell function (see
below) (Fig. 1).
© 2012 The Authors.
Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology
Calcineurin regulates gonadotrophin gene expression via
nuclear factor of activated T-cells (NFAT) and Nur77
NFAT is the most extensively studied calcineurin target; first identi-
fied in T-cells, it is rapidly induced to activate the IL-2 promoter.
However, it is widely expressed and plays extensive roles in regulat-
ing gene expression for diverse aspects of cell function. Four NFAT
isoforms have been identified, NFAT1 (NFATc2), NFAT2 (NFATc1),
NFAT3 (NFATc4) and NFAT4 (NFATc3), all of which are comprised of
an N-terminal transactivation domain, a DNA-binding domain and
a NFAT regulatory region. This regulatory region is phosphorylated
on multiple serines by various kinases, including casein kinase 1
and glycogen synthase kinase 3, which act to repress NFAT activity
by keeping it in the cytoplasm. NFAT is activated by calcineurin-
mediated dephosphorylation of these same residues, allowing the
NFAT to translocate to the nucleus to activate transcription of its
target genes. In both the cytoplasm and nucleus, NFAT interacts
with a variety of other transcription factors, which include AP-1,
GATA4, Foxp and MEF2, and these interactions play a crucial role in
determining the specificity of the NFAT-binding to its target genes.
Nuclear NFAT is inactivated by phosphorylation, which causes it to
return to the cytoplasm (58,59).
In aT3-1 cells, NFAT 2, 3 and 4 are expressed, with NFAT3 being
notably translocated into the nucleus, in a calcineurin-dependent
manner, after GnRH treatment (50) (Fig. 1). In these cells, NFAT 2
and 4 appear to be constitutively nuclear, although, in studies in
which NFAT2-EFP was transfected into LbT2 cells, the over-
expressed protein did oscillate between cellular compartments in
response to GnRH (57). NFAT3 is required for GnRH-induced aGSU
expression in the aT3-1 cells, whereas NFAT4 may play a role in its
basal expression. In support of a role for NFAT3 in mediating the
effect of GnRH on aGSU gene expression, the calcineurin-respon-
sive region of the promoter maps to a sequence ( 420 to
346 bp upstream of the tss) already shown to mediate both the
GnRH and calcium responses, and this common sequence
(tTTCCTGTT) contains a consensus NFAT-binding element (50,60,61).
Recently, calcineurin-driven NFAT was also reported to mediate
GnRH-induced expression of the early response genes, Jun and
Aft3. It was shown that NFAT binds the Jun promoter and interacts
with Jun through AP-1 sites (62). Given that AP-1 sites also regu-
late aGSU expression, this suggests an additional mechanism of the
role of NFAT with respect to transducing the effect of GnRH to this
gene.
NFAT also regulates FSHb gene transcription, although this effect
is indirect, mediating GnRH stimulation of the Nur77 transcription
factor. The expression of the Nur77 is elevated by GnRH in a potent
and rapid manner, and this is calcineurin-dependent (26,50,52,63)
(Fig. 1). In T-cells, the transcription of Nur77 occurs via calcineurin-
activated NFAT, which interacts with MEF2D at the Nur77 gene
promoter (64). Also in the gonadotroph, NFAT3 is recruited to the
Nur77 promoter after GnRH treatment concomitant with the asso-
ciation of RNAPII, and NFAT appears to play a central role because
its knockdown reduced the GnRH stimulatory effect on Nur77
expression. In the aT3-1 cells, Nur77 over-expression is sufficient to
de-repress expression of the FSHb gene, whereas FSHb promoter
Journal of Neuroendocrinology, 2012, 24, 1463–1475

activity and transcript levels are reduced after NFAT3 knockdown in
LbT2 cells, suggesting that NFAT3-activated Nur77 is required for
GnRH-induction of the FSHb gene (26,50).
Apart from the involvement of calcineurin in the GnRH stimula-
tion of Nur77 expression, it is likely also involved in the GnRH-acti-
vated dephosphorylation of Nur77 at S354 (Fig. 1). Mutation of this
residue indicates that the dephosphorylation is a means of activat-
ing Nur77 transcriptional activity (26). This is also the mechanism
of Nur77 activation by adrenocorticotrophic hormone, involving
dephosphorylation at the same residue, although, unlike at the
FSHb gene, the phosphorylation prevented DNA binding in the con-
texts tested (65). Although we have yet to determine the function
of this dephosphorylation in FSHb gene activation, Nur77 is known
to interact with SMRT, which represses expression of the FSHb gene
in these cells (26,66,67). It is therefore quite possible that the
dephosphorylation causes a switch in Nur77 interacting proteins,
allowing it to dissociate from SMRT and recruit coactivators.
A role for calcineurin-regulated Transducer of Regulated
CREB (TORC)
The TORCs are transcriptional coactivators whose activity is regu-
lated by calcineurin-mediated dephosphorylation. Also known as
CREB-Regulated Transcription Coactivators, this group of proteins
was originally identified as interacting with, and potently increasing
the activity of CREB, however, they are now known to coactivate
additional transcription factors, including c-jun, c-fos, ATF1 and
ATF4 (68,69). Unlike the CREB coactivation by CBP, the actions of
TORC are independent of CREB phosphorylation at Ser 133,
although TORC appears to coactivate at only a subset of CREB tar-
gets (70,71). Dephosphorylation of the TORC proteins by calcineurin
allows their nuclear import and thus their transcriptional activity.
Conversely, they are negatively regulated by phosphorylation, for
example by protein kinase A-induced salt-inducible kinase, which
prevents their nuclear localisation (72,73).
Although enriched in the brain and lymphocytes, TORC proteins
are expressed quite ubiquitously at low levels, and we were able to
detect TORC1 and TORC3 proteins (but not TORC2) in the aT3-1 cells
(Melamed, unpublished data). TORC1 over-expression was sufficient
to increase aGSU promoter activity, whereas its knockdown indicated
that TORC1 plays an essential role in GnRH-activated aGSU tran-
scription. However, this gene is not regulated by CREB, and TORC
does not appear to be recruited to the aGSU promoter by CREB.
Because we were unable to discern synergistic effects of TORC with
any transcription factor in the activation of this gene, the factor
responsible for recruiting TORC remains to be identified, although it
may already be found at the promoter in untreated cells (50).
CREB clearly does play a role in GnRH-mediated activation of
the FSHb gene (74), and it acts synergistically with TORC1 (50). The
CREB-TORC1 synergy is further enhanced by Nur77, although
Nur77 and TORC also exert a powerful synergistic effect without
the addition of CREB. TORC1 over-expression also increased the
effect of GnRH, although its knockdown revealed more of an effect
on basal promoter activity than on that induced by GnRH. Notably,
the GnRH-induced phosphorylation of CREB at S133 prevents its
Journal of Neuroendocrinology, 2012, 24, 1463–1475
Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology
GnRH signalling downstream of calmodulin 1467
interaction with TORC1, presumably favouring the recruitment of
CBP (74). Given that this serine is required for the full effect of
GnRH but not for that of TORC1, TORC1 may not be required for
the GnRH-stimulated FSHb promoter activity. However, TORC1 does
appear to play a role in regulating FSHb gene expression because
TORC1 knockout male mice have significantly lower circulating FSH
levels than their wild-type counterparts; although this was not seen
in female mice, which were tested only at pro-oestrous (75). The
role of TORC in FSHb gene expression has thus to be fully eluci-
dated, although it might provide a different mechanism of activat-
ing transcription as part of an alternatively regulated GnRH-
independent pathway, at the same time as still allowing for cross-
talk with the GnRH-activated cascade.
Although calcineurin is known to promote TORC nuclear localisa-
tion, and GnRH treatment causes dephosphorylation of TORC1 in a
calcineurin-dependent manner, the levels of nuclear TORC1 were
not increased by GnRH treatment. TORC1 appears to be inherently
unstable in these cells and, after GnRH is added, the existing TORC1
is rapidly degraded. Subsequently, the newly synthesised protein
appears to have a protected N-terminus which is associated with
its enhanced activity (50). Instability of the TORC1 before GnRH
treatment is a result of the actions of calpain and the proteasome,
and we have proposed that the degradation of the newly synthes-
ised TORC is regulated by GnRH actions on the calpain-calpastatin
system, possibly through ERK-activated phosphorylation. This is
similar to the regulation of PKCe degradation in rat pituitary GH4C1
cells by thyrotrophin-releasing hormone (76). However, other expla-
nations are also quite possible.
Wide-ranging effects of calcineurin via dephosphorylation
of Pin1
We recently reported a novel target of calcineurin in the gonado-
trophs: the ubiquitously expressed peptidyl-prolyl isomerase, Pin1
(49). Pin1 is the only known enzyme that binds a phosphorylated
serine or threonine preceding a proline (pSer/Thr-Pro) and isomeris-
es the cis-trans conformation of the prolyl-peptidyl bond. It con-
sists of a short N-terminal WW domain that binds only to pSer/
Thr-Pro motifs, providing target specificity, and a C-terminal cata-
lytic domain that isomerises the prolyl-peptidyl bond. Pin1 thus
mediates a phosphorylation-induced conformational change in the
target protein, which leads to altered activity, providing additional
insight into the role of this post-translational modification (77,78).
Notably, Ser/Thr-Pro is a common motif that is phosphorylated by
various kinases, particularly by the MAPKs which form an integral
part of GnRH-induced signal transduction, and Pin1-catalysed prolyl
isomerisation has been shown to be involved in a wide variety of
contexts including the regulation of transcription, protein–protein
interaction and/or protein subcellular localisation (77,78).
Pin1 activity is tightly regulated via several mechanisms that
determine its ability to bind the substrate, its catalytic activity and/
or its cellular localisation (Fig. 1). Phosphorylation of Pin1 at S16,
in the pSer/Thr-Pro binding pocket, is a key mechanism of control
because it prevents the WW domain from interacting with pSer/
Thr-Pro motifs on its substrates and may facilitate Pin1 nuclear
© 2012 The Authors.
1468 P. Melamed et al.
export (79). In the gonadotrophs, intriguingly, GnRH increases levels
of phosphoPin1, as does forskolin or, to a lesser extent, phorbol
12-myristate 13-acetate (PMA) treatment. This suggests a role for
GnRH-activated PKC, or protein kinase A which has been implicated
in Pin1 phosphorylation in the past (79). Conversely however, Pin1
and calcineurin co-precipitate and this interaction increases after
GnRH treatment. The GnRH-activated calcineurin acts to dephos-
phorylate Pin1, thus keeping it in its active state; this may be cru-
cial to counteract the apparently paradoxical increase in Pin1
phosphorylation after GnRH treatment (49).
One of the targets of Pin1 in the gonadotrophs is the transcrip-
tion factor Sf-1. Pin1 facilitates the phosphorylation-to-ubiquitina-
tion transition of Sf-1 (Fig. 1), which is required for Sf-1
interaction with Pitx-1; this interaction is essential for the synergis-
tic transcriptional activation of the LHb gene (49,80). However, Sf-1
is required for the full GnRH response of all three gonadotrophin
subunit genes, while many of the other GnRH-activated transcrip-
tion factors that regulate gene expression in the gonadotrophs also
contain pSer/Thr-Pro motifs and are activated through phosphoryla-
tion by MAPKs, making them likely Pin1 targets.
In other contexts, Pin1 has been shown to bind and regulate the
activity of additional modulators of gonadotrophin gene expression,
including ERa, c-Jun, b-catenin, SMADs 2 and 3, SMRT, NFAT and
TORCs (2,3,43,44,50,78,81-84). For NFAT and the TORCs, the effect
of the Pin1 is to repress transcriptional activity of the factor by
keeping it in the cytoplasm; moreover, the interaction of NFAT with
Pin1 specifically prevents NFAT activation by calcineurin (81,83).
Although the role of Pin1 in regulating NFAT and/or TORC activity
in the gonadotrophs has yet to be verified, these studies indicate
that cytoplasmic, presumably phosphorylated, Pin1 also plays a role
in determining the signalling pathways to differential gene expres-
sion, and may help contribute an explanation for the GnRH-induc-
tion of Pin1 phosphorylation. This paradoxical regulation further
highlights the important role of calcineurin in the regulation of
Pin1 function by phosphorylation/dephosphorylation and indicates
that Pin1 plays a likely widespread role not only in the gonado-
troph, but also in MAPK signalling cascades in more diverse
contexts.
Effects of GnRH via Ca2+/CaM signalling on the cell cycle
and gonadotroph proliferation
Ca2+/CaM signalling plays a variety of roles in the regulation of the
cell cycle, and CaM levels vary through the cell cycle, increasing
just before the S-phase. CaM over-expression accelerates prolifera-
tion, apparently due to a shortening of progression through G1,
whereas, conversely, a reduction of CaM levels inhibits cell prolifer-
ation (25). We have already shown that GnRH induces an increase
in the number of aT3-1 cells (6) and we now report preliminary
experiments showing that this effect is abolished by treatment with
cyclosporin A (Fig. 2A), indicating that it is likely mediated primarily
by the Ca2+/CaM activation of calcineurin.
It has been suggested that Ca2+ and CaM are required for the
activation of cyclin D1/cdk4 and pRb hyperphosphorylation and,
indeed, we show here in a pilot study that GnRH increases the
© 2012 The Authors.
Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology
expression of cyclin D1, although not cdk4 (Fig. 2B,C). Paradoxically,
the cdk4 promoter is reportedly inhibited by calcineurin-regulated
NFATc2, while calcineurin is also able to dephosphorylate cdk4
directly, leading to its inactivation (85,86). GnRH-induced NFAT
activity does not appear to play a similar role in GnRH-activated
proliferation of the immature gonadotrophs because these initial
studies reveal that cdk4 expression is reduced by cyclosporin A but
not affected by GnRH (Fig. 2C). Any role for GnRH-activated post-
translational regulation of cdk4 activity via calcineurin remains to
be investigated.
GnRH likely also affects cell cycle progression via its calcineurin-
mediated dephosphorylation of Pin1, which regulates transcription
of cyclin D1, as well as the accumulation of both cyclin D1 and
b-catenin in the nucleus (78,87–89). Pin1 stimulates cyclin D1 gene
expression via enhancing the activity of JNK, as well as stabilising
c-jun, both of which are activated downstream of GnRH in a man-
ner that is dependent on b-catenin (4,52,82,90–92). GnRH and Pin1
have been shown to stimulate translocation of b-catenin to the
nucleus (91,93), and we show here in a novel study that b-catenin
plays a role in GnRH-induced cyclin D1 gene expression, as well as
its induction of the proliferation of aT3-1 cells (Fig. 2D–E).
Additional anti-proliferative effects of calcineurin-inhibition, as
shown in various cell types, are mediated via growth factor-medi-
ated increases in p21 and/or the accumulation of p27, which are
both cyclin-dependent kinase inhibitors (CKIs) that prevent activa-
tion of the cyclin/cdk complexes (25,94). In our preliminary studies,
GnRH treatment led to a marginal increase in p27 and p21 levels
in aT3-1 cells, and this was inhibited by cyclosporin A treatment
(Fig. 2F-G), suggesting an involvement of calcineurin in their tran-
scription, as seen previously in other cell contexts (95,96). At low
levels, these CKIs may not suffice to inactivate the cyclin/cdk com-
plex and may actually promote their activation through facilitating
assembly of the complexes, increasing their nuclear import and by
stabilising cyclin D (25).
In LbT2 cells, GnRH no longer induces cell proliferation but reduces
cell numbers through inhibiting proliferation and/or inducing apopto-
sis (1,6). GnRH is seen to have anti-proliferative/pro-apoptotic effects
in a variety of other cells as well, although, for the most part, the
mechanisms have either not been elucidated or appear to vary
between the different cell types (97–100). We are currently investi-
gating the mechanisms through which GnRH reduces cell number of
the LbT2 cells, and the reasons for the apparent shift in response with
differentiation. However, GnRH was reported to increase p21 mRNA
levels quite significantly (three- to six-fold) in LbT2 cells (4,63,101),
suggesting that this CKI induction might play a role in blocking cell
cycle progression. GnRH was also shown to reduce expression of a
number of the cyclins, cyclin-associated kinases and several subunits
of the anaphase-promoting complex (4). Although these cells were
immortalised using the SV40 large T antigen, which could potentially
alter their proliferative response, the vast majority of large T-respon-
sive genes are not affected by GnRH and/or activin treatments (4),
indicating that the presence of SV40 large T antigen is unlikely to
confound these conclusions. Confirmation of this effect of GnRH in
primary partially-differentiated embryonic gonadotroph cells in
embryos is technically difficult, however GnRH was seen to increase
Journal of Neuroendocrinology, 2012, 24, 1463–1475
 GnRH signalling downstream of calmodulin 1469

(A) (B) (C) Cdk4

1.8 GnRH (h) 0 3 5 8 1.20

treated (normalised to RPLPO)

C B Control

mRNA levels fold over non

1.6 B GnRH
Absorbance (fold over

Cyclin D1 1.00
nontreated control)

1.4 Control
1.2 B 0.80
GnRH β -actin
1 A A
0.60
0.8
0.6 0.40
A A
0.4 0.20
0.2
0 0.00
NT CSA
(D) NT CSA
(E) 2
GnRH: – + – + B
1.8 Control
siRNA: GFP GFP β -catenin β -catenin siRNA: GFP β -catenin GnRH
1.6

Absorbance (fold over

β -catenin

nontreated control)
Cyclin D1 1.4
A
GAPDH 1.2
β -actin A
1
A
Normalised 1 2.93 2.79 1.28 1.06 1.34 1.22 0.8
values 0.6

(F) p27 (G) p21 0.4

B 2.4 B Control 0.2
Control
mRNA levels fold over nontreated

1.4
mRNA levels fold over nontreated

GnRH 0
A GnRH
1.2 2.0 siGFP siβ -catenin
(normalised to RPLPO)
(normalised to RPLPO)

A
1.0 A 1.6 A
0.8 A
1.2 A
0.6
0.8
0.4
0.2 0.4

0.0 0.0
NT CSA NT CSA

Fig. 2. Calcineurin plays a role in gonadotrophin-releasing hormone (GnRH)-activated aT3-1 cell proliferation. (A) An XTT assay to assess cell numbers was car-
ried out in aT3-1 cells after some were exposed to GnRH for 24 h, with or without 30 min cyclosporine A (CsA) pretreatment. Absorbance is expressed relative
to that in nontreated (NT) cells without GnRH; mean ± SEM (n = 7–9). ANOVA followed by a Bonferroni t-test compared all means and those that are similar
(P > 0.05) are designated the same letter. (B) Cyclin D1 protein levels (with b-actin as internal control) were assessed by western analysis in aT3-1 cells treated
with GnRH (10 nM) for 0–8 h. (C) Real-time quantitative polymerase chain reaction (PCR) analysis was performed to measure the effects of GnRH (8 h) with
and without CsA pre-treatment (10 min before GnRH) on mRNA levels of Cdk4. Transcript levels were quantified using a standard curve and are represented
relative to levels in untreated controls, after normalisation to levels of large ribosomal protein (RPLPO). Mean ± SEM (n = 3–4); statistical analysis as in Fig. 2(A).
(D) Small interfering (si)RNA targeting b-catenin, or green fluorescent protein (GFP) as control (both in pSUPER) was transfected 48 h before GnRH treatment
(3 h), after which RNA was extracted, reverse transcribed and PCR amplification carried out for cyclin D1 and b-actin. PCR products were resolved in agarose
gel and the intensity of each band quantified relative to untreated siGFP-control, after normalisation with b-actin levels. Western analysis for the b-catenin
knockdown is also shown. (E) The siRNA targeting b-catenin was transfected and, after 40 h, some cells were treated with GnRH (20 nM for 16 h) before a
bromodeoxyuridine assay was performed to assess cell proliferation. Absorbance is expressed and the data are presented as in Fig 2(A); mean ± SEM (n = 4).
(F, G) Real-time quantitative PCR analysis was carried out as in Fig. 2(C) to assess effects of CsA and/or GnRH on mRNA levels of (F) p27 and (G) p21. Experimental
details and data presentation are as in Fig. 2(C). All experiments were carried out on at least three independent occasions and representative results are shown.

gonadotrope proliferation in adult mice and rats under certain cir- cytoplasm, allowing activation of the caspases and also of the nuc-
cumstances, including at specific stages of the oestrous cycle or after leases required for the apoptotic response (16,105–107). However,
castration (102–104), suggesting a latent ability to proliferate in other forms of cell death triggered by Ca2+-mobilisation are CaM-
response to GnRH that can be moderated by other factors including dependent, including the activation of death-associated protein
steroids. kinase and AMPK, which are involved in autophagic cell death
Calcineurin is clearly not the only way in which calcium influx (108). AMPK is activated in gonadotrophs in response to GnRH,
stimulates an apoptotic response, which is driven in many cases although neither protein has yet been linked to cell death after
by CaM-independent mechanisms via caspase activation. The GnRH treatment (29). A specialised form of apoptosis, anoikis,
Ca2+-influx into the mitochondria increases cytochrome C in the involving detachment of cells from their surrounding matrix, is also

Journal of Neuroendocrinology, 2012, 24, 1463–1475 © 2012 The Authors.

Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology
1470 P. Melamed et al.

regulated by Ca2+-CaM, in which changes in Ca2+ homeostasis lead
to the activation of Rho kinase signalling (108). Although the
induction of this form of apoptosis by GnRH has yet to be
reported, GnRH does induce major cytoskeletal rearrangement and
gonadotroph cell migration, likely involving Rho signalling cascades
(109,110). GnRH-induced Rho signalling may be regulated in part
by a small GTP-binding protein, Gem, which is activated by GnRH
and regulated by CaM, and was shown, in other contexts, to bind
Rho kinase to alter downstream signalling (111).
A role for calmodulin in moderating GnRH-induced
signalling through negative-feedback?
Gem and its regulation by GnRH
Gem belongs to the RGK (comprising Rad, Gem and Kir, as well as
Rem1 and Rem2) subfamily of small Ras-related GTP-binding
proteins that moderate various G-protein coupled pathways. These
differ structurally from other GTPases in that they do not contain
lipid modification for anchorage to the membrane. Gem and other
members of this group have several roles, including inhibiting Ca2+
channels, while Gem also affects rearrangement of the cytoskeleton
through altering Rho signalling. The activity and localisation of
Gem are regulated through CaM and 14-3-3 proteins (111,112).
Gem is expressed at higher levels in aT3-1 than in LbT2 cells,
while in the LbT2 cells, its mRNA levels increase after GnRH treat-
ment (4,6,52). Given that Gem inactivates the L-type VSCC that are
utilised by GnRH for Ca2+ influx, it was suggested that Gem might
reduce external calcium entry as part of a negative-feedback loop,
so controlling pathways downstream of calcium (8,52,113). Consis-
tent with this, we found that Gem, which is initially found
throughout aT3-1 cells, moves out of the nucleus after exposure to
GnRH for 15–30 min (Fig. 3A). This effect appears to be indepen-
dent of CaM because a mutant Gem that cannot bind CaM
(W269G) (113) translocated to the cytoplasm in a manner similar
to the wild-type protein (Fig. 3A). The export is likely therefore

(A) (B)
WT Gem HDAC7 W269G Gem HDAC7 6 Control a
Firefly luciferase activity (fold over +GnRH
Before 5
normalised to Renilla)
nontreated control,
GnRH b
4
c

15 min 3
GnRH
2
A A B
30 min 1
GnRH
0
GSU Gem WT Gem W269G

(C) siGem
4 Control D – +
Firefly luciferase activity (fold over

Gem
3.5 +GnRH
Tubulin
normalised to Renilla)
nontreated control,

3
2.5 C

2
1.5 B
1
A
0.5
0
GSU + siGem

Fig. 3. Gonadotrophin-releasing hormone (GnRH)/calmodulin (CaM)-regulated Gem in gonadotrophs. (A) aT3-1 cells plated on glass cover-slips were co-trans-
fected with expression vectors for yellow fluorescent protein-tagged histone deacetylase (HDAC) 7 together with enhanced green fluorescemt protein (EGFP)-
tagged wild-type (WT) Gem (first and second columns) or EGFP-tagged W269G Gem mutant (third and fourth columns). After 48 h, GnRH in phenol-red free
medium was added. Confocal images of cells were captured initially and after 15 and 30 min. A single representative cell is shown for each treatment/time
point; scale bars = 10 lm. (B) aT3-1 cells were transfected with mouse glycoprotein hormone a-subunit promoter-luciferase reporter gene (aGSU), and WT
Gem or the W269G mutant were overexpressed, and samples exposed to GnRH treatment (100 nM, 8 h). After 60 h, luciferase assays were performed, and lev-
els normalised to those of Renilla; the results are expressed as the fold induction over the mouse aGSU-luciferase control-transfected and untreated cells. Sta-
tistical analysis (ANOVA followed by Bonferroni t-test) compared mean values separately for untreated (upper case) and GnRH-treated (lower case) cells, and
those designated by different letters are significantly different (P < 0.05); mean ± SEM (n = 5–6). (C) aT3-1 cells were transfected with mouse aGSU-luciferase
(as above) and some with small interfering (si)RNA targeting Gem (in pSUPER). Some of the cells were treated with GnRH, luciferase assays performed, values
normalised and presented all as above (n = 6). All experiments were carried out on at least three independent occasions and representative results are shown.
Insert: western blot showing levels of Gem in aT3-1 cells 60 h after transfection of siGEM.

© 2012 The Authors. Journal of Neuroendocrinology, 2012, 24, 1463–1475

Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology

driven by GnRH-activated phosphorylation of Gem, which facilitates
recognition by 14-3-3 proteins and can be targeted by a number of
kinases, as reported in other cell contexts (111,114,115). Both
groups of cells also underwent morphological changes after expo-
sure to GnRH (Fig. 3A), which has already been reported in these
cells (5), and is in keeping with actions of Gem on cytoskeletal
rearrangement (115).
Although GnRH regulates Gem localisation, our preliminary stud-
ies did not provide any evidence of Gem exerting a negative effect
on gonadotrophin gene expression. GnRH-induced aGSU promoter
activity in aT3-1 cells is highly dependent on calcium influx (60),
yet, in our preliminary studies using aGSU-promoter-driven lucifer-
ase assays, over-expression of wild-type Gem did not reduce
GnRH-stimulated promoter activity but, instead, had a minor stimu-
latory effect, although the W269G mutant reduced promoter activ-
ity by approximately 30% (Fig. 3B). Furthermore, small interfering
RNA-mediated partial knockdown of Gem also had an inhibitory
effect on the promoter activity (Fig. 3C). Taken together, these
results indicate that Gem does not exert negative-feedback on the
effect of GnRH but may perhaps play a novel positive role, possibly
being regulated through its interaction with CaM in the cytoplasm.
RGS proteins
RGS proteins, which act as GTPase-activating proteins of Ga,
including Gaq that transduces much of the effect of GnRH, have
also been implicated in modulating GnRH-induced signalling and
these too are regulated by CaM. RGS2 expression is increased by
GnRH and it was proposed that the ability of RGS2 to down-regu-
late Gaq activity might contribute to the frequency-dependent
downstream gene activation (8,52). In keeping with this, RGS2
over-expression in aT3-1 cells reduced GnRH signalling via Gaq/11,
resulting in a marked reduction in phospholipase C activation, while
also decreasing GnRH-induced mRNA levels of Egr-1 and c-fos
(116,117).
Although RGS repression of Gaq/11 can be inhibited by various
kinases including PKC, as well as by phosphatidylinositol-3,4,5,-tris-
phosphate (PIP3), it is restored by Ca2+/CaM (118,119). It was sug-
gested that, in cardiac myocytes, the cyclical inhibition of Gaq/11 is
determined by CaM-stimulated RGS4, which inactivates Gaq/11
leading to a reduction in the formation of PIP3; this results in a
drop in Ca2+ levels that leads to CaM dissociation from RGS4, after
which PIP3-mediated repression of RGS4 is restored and once more
inhibits Gaq/11 (118,120). Such a role for RGS2 in negative-feed-
back during GnRH-induced signalling in the gonadotroph appears
feasible, although this has yet to be demonstrated.
Ca2+/CaM-sensitive GnRHR-ERK signalling complexes
RGS proteins are localised in membrane-associated lipid rafts where
they interact with various scaffold proteins to concentrate these
proteins with the G-protein coupled receptor for regulation of
downstream signalling, and disruption of these rafts was seen to
inhibit the interaction between RGS4 and CaM (118,121–123). In the
gonadotrophs, compartmentalisation of a putative Ca2+-sensitive
Journal of Neuroendocrinology, 2012, 24, 1463–1475
Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology
GnRH signalling downstream of calmodulin 1471
signalling complex into lipid rafts together with the GnRHR was
proposed to integrate Ca2+ and ERK signalling and poise them for
the GnRH-activated response. Both CaM and 14-3-3b colocalise in
these membrane-associated lipid rafts together with the GnRHR, Gq
and c-Raf kinase, and it was suggested that CaM serves as a link
between the VSCC, Ca2+ and ERK (13,53). Indeed, inhibition of CaM
blocks GnRH- induced ERK1/2 but not JNK activation in aT3-1 cells,
and it also attenuated ERK-dependent gene reporter activity of c-
fos, aGSU and MAPK phosphatase-2 promoters, in a manner that is
at least partially CaMK-independent (53). Although RGS2 has yet to
examined in the context of these gonadotroph lipid rafts, it appears
likely that it is part of this signalling complex, being localised and/or
regulated in the rafts by CaM, to provide a mechanism of Ca2+-
sensing in GnRH-induced ERK signalling.
Additional signalling complexes associated with ERK-activation
by GnRH are expected also to be regulated by CaM-activated path-
ways (12,13,124–127). GnRH stimulates the rapid assembly of an
ERK-activating complex that contains the proline-rich tyrosine
kinase, Pyk2, which serves as a scaffold for c-Src, Grb2 and Sos
and is essential for ERK-dependent gene transcription. The catalytic
domain of Pyk2 interacts directly with Ca2+-CaM, and this is
required for its phosphorylation after GnRH exposure, as well as its
functional activity. The transmission of the ERK-activating signal to
the nucleus by the Pyk complex appears to be via focal adhesion
complexes and is dependent on a functional actin cytoskeleton. Its
role in ERK activation and translocation to the nucleus led to the
suggestion that this complex also may form a link between VSCC
Ca2+ signals and the ERK activation pathway (12,13,125,126).
The GnRHR, c-Src, and focal adhesion kinase (FAK) are found
localised together in an additional signalling complex, likely also in
lipid rafts, that is restructured upon GnRH treatment. It was pro-
posed that this complex acts to restrict the amount of active ERK
from moving to the nucleus, and suggested that the ERK might
direct phosphorylation of FAK and paxillin to mediate some of the
GnRH-induced changes in cell morphology (127,128). GnRH has
been shown to promote cytoskeletal rearrangement, cell morphol-
ogy and adhesion, and Rho-kinase pathways were implicated in this
effect (110). It is an interesting speculation that Gem, which is reg-
ulated by GnRH and known to alter cell morphology by affecting
Rho kinase substrate specificity, might play a role in this complex.
Conclusions and future directions
GnRH has far-reaching effects on the gonadotrophs beyond merely
inducing release and synthesis of the gonadotrophin hormones, and
CaM-driven pathways, which are activated in response to increases
in Ca2+, clearly play a major role in many of these effects, as high-
lighted in this review. Although we have focused on recent literature
describing the effects of calcineurin and CaMKs, this is likely only a
subset of the pathways activated downstream of CaM in response to
GnRH. Hundreds of proteins are known to be bound by CaM, includ-
ing various DEAD/H box proteins and enzymes involved in protein
ubiquitination and degradation, and there is particularly strong
evidence for its dominant cytoplasmic roles in both signalling and
vesicular trafficking (129–131). However, CaM also translocates to
© 2012 The Authors.
1472 P. Melamed et al.
the nucleus in response to various stimuli, and although its nuclear
functions have barely been studied, it appears to be involved in sig-
nalling to transcription; in neuronal cells, it was suggested to provide
a cellular ‘memory’ in facilitating repeated neural activity (132).
Moreover, a novel role for CaM was demonstrated recently, in which
it binds the signal peptides of small secretory proteins, and acts as a
chaperone for their translocation through the cytosol and into the
endoplasmic reticulum (133). We have likely only just begun to
understand the role of CaM in GnRH signalling in the gonadotroph.
Acknowledgements
This research was supported by the Technion VPR fund-Mallat family
research fund, and the European Framework programme Marie Curie
Actions.
Received 15 March 2012,
revised 24 June 2012,
accepted 3 July 2012
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