g-protein-coupled receptor signaling and the egf network in endocrine systems

G-protein-coupled receptor signalingand the EGF network in endocrinesystemsMinnie Hsieh and Marco Conti

Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University, Stanford, CA 94305-5317, USA

The epidermal growth factor (EGF) network is com-

posed of a complex array of growth factors synthesized

as precursors and expressed on the cell surface. These

latent growth factors are activated by cleavage and

shedding from the cell surface and act by binding to

various homo- and hetero-dimers of the EGF receptors

(ErbBs). Although the exact molecular steps are poorly

understood, ligand binding to G-protein-coupled

receptors as diverse as the b-adrenoceptors or the

lysophosphatidic acid receptors leads to shedding of

EGF growth factors and activation of EGF receptors.

Recent observations from the pituitary and in the ovary

are providing new insight into the role of this network in

endocrine systems.

Introduction

Given the array of extracellular cues to which they areexposed, cells have developed complex machinery for thereception and interpretation of external stimuli. Multipleintracellular signaling pathways are required to receivethese signals and translate them into changes in cellularfunctions. A common theme in the arrangement of thesepathways is the integration and crosstalk betweencontiguous cascades to fine tune cellular functions asdiverse as cell proliferation, differentiation and migration.The transactivation of receptor tyrosine kinases byG-protein-coupled receptors (GPCRs) is one example ofcommunication and cooperation between different signal-ing networks. In a variety of cellular contexts, agonistbinding to GPCRs results in transactivation of theepidermal growth factor (EGF) receptor (EGFR; alsoknown as ErbB1 or HER) and activation of the extra-cellular signal-regulated kinase/mitogen-activated pro-tein kinase (ERK/MAPK) cascade. Traditionally, theEGF network has been viewed as a major orchestrator ofcell replication under physiological and pathologicalconditions. However, this signaling network has muchbroader functions in the body, for instance in earlydevelopment and in organogenesis, and in tissue homeo-stasis in the adult. Here, we review evidence supportinginvolvement of the EGF network in the regulation ofendocrine and reproductive processes. These studies,together with those examining GPCR-mediated EGFR

Corresponding author: Conti, M. ([email protected]).Available online 28 July 2005

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transactivation, provide new insight into the mechanismby which signals arising from GPCRs can be transducedvia the EGF network to affect crucial endocrine functions.

GPCR signaling to the ERK/MAPK pathway

GPCRs are the largest family of cell surface receptors,with more than 1000 genes encoding this class of proteins.They are characterized by a conserved structural motif ofseven transmembrane-spanning regions, and undergoagonist-induced conformational changes that expose intra-cellular binding sites for the heterotrimeric GTP-bindingproteins (G proteins) (reviewed in [1]). Interaction ofGPCRs with G proteins leads to the exchange of GDPfor GTP in a binding pocket of the Ga subunit, followedby dissociation of G-protein complexes into activateda subunits and bg dimers. These, in turn, act on effectormolecules to promote second messenger-generating sig-naling cascades, including the cAMP-, inositol trisphos-phate- and Ca2C-dependent pathways. Signaling throughthese pathways usually culminates in the activation ofkinases and changes in protein phosphorylation. Some ofthese kinases are directly regulated by second mes-sengers, but many constitute modules further down-stream of the signaling cascade, such as the RAF–MAPKkinase–ERK (RAF–MEK–ERK) module.

The mechanism by which GPCRs activate the ERK/MAPK cascade is complex and often cell specific. Some ofthe biochemical steps linking GPCRs to MAPK activationinclude the kinases protein kinase A (PKA) [2,3], proteinkinase C (PKC) [4] or Src/Pyk2 [5,6], and might involveregulation of small G proteins and guanine nucleotideexchange factors [(e.g. EPAC (exchange protein directlyactivated by cAMP)] [7,8]. In other instances, more elabor-ate circuits are involved in activation of this signalingmodule. Daub et al. [9] were the first to demonstrate thattheGPCRligands lysophosphatidicacid (LPA), endothelin-1and thrombin induced rapid tyrosine phosphorylation ofthe EGFR and ErbB2 in Rat-1 fibroblasts, and that thespecific EGFR inhibitor tyrphostin AG1478 or a dominant-negative EGFR mutant abrogated GPCR-induced ERK/MAPK activation.

Further studies confirmed that many GPCRs utilizeEGFR transactivation to couple to the ERK/MAPKcascade in diverse cell types (reviewed in [4,10]). Forexample, it has been proposed that activation of theb2-adrenoceptor in COS7 cells induces receptor complex

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Review TRENDS in Endocrinology and Metabolism Vol.16 No.7 September 2005 321

formation with the EGFR, with subsequent EGFRtransactivation and MAPK activation [11]. Stimulationof head and neck squamous cell carcinoma (HNSCC) cellsby the GPCR agonists LPA, carbachol, bradykinin orthrombin causes the rapid tyrosine phosphorylation ofEGFR, illustrating the convergence of different activatingsignals on a common pathway [12]. As in other cells, EGFRtyrosine kinase activity was required for LPA-inducedMAPK activation, and inhibition of EGFR signalingabrogated LPA-stimulated proliferation and migration ofHNSCC cells. The pathophysiological relevance of thisregulation is underscored by the finding that activation ofrat neonatal cardiomyocytes by phenylephrine, angio-tensin II and endothelin-1, known inducers of cardiachypertrophy, resulted in enhanced EGFR tyrosine phos-phorylation [13]. The discovery that GPCR-inducedcardiac hypertrophy was attenuated by a specific inhibitorof ADAM12 (a disintegrin and metalloprotease 12), whichis involved in processing of one of the EGFR ligands(discussed below), further established the requirementfor crosstalk in mediating pathophysiological events.Additional GPCR ligands shown to mediate EGFR trans-activation and stimulation of the ERK/MAPK pathwayinclude oxytocin [14] and prostaglandin F2a [15,16],raising the possibility that this circuit operates inreproductive and endocrine systems.

EGF signaling network

The ErbB receptor family comprises four members: EGFR(ErbB1, HER), ErbB2 (Neu, HER2), ErbB3 (HER3) andErbB4 (HER4) [17,18]. These receptors have in common acysteine-rich extracellular domain, a single membrane-spanning region and a cytoplasmic domain containing atyrosine kinase domain and tyrosine autophosphorylationsites. Ligand binding to specific ErbB receptors promotesreceptor homo- or heterodimerization, activation of theintrinsic tyrosine kinase, tyrosine phosphorylation ofseveral substrates and the ErbB receptor itself, andstimulation of downstream signaling cascades [19].

ErbB receptors are activated by members of theEGF-like growth factor family. Each EGF-like growthfactor contains an EGF-like motif characterized by sixcysteine residues that form three intramolecular disul-fide-bonded loops, and that confer binding specificity forthe ErbB receptors [17,18]. Group 1 ligands bind speci-fically to EGFR, and consist of EGF, transforming growthfactor a (TGF-a) and amphiregulin. The neuregulins thatcomprise group 2 bind ErbB3 and ErbB4. Group 3members include heparin-binding EGF-like factor(HB-EGF), betacellulin and epiregulin, and bind bothEGFR and ErbB4. Although ErbB2 has no known ligand,it heterodimerizes with other ErbB receptors to form apotent signaling complex. ErbB3 that has impaired kinaseactivity only becomes phosphorylated and forms an activereceptor complex when partnered with another ErbBreceptor.

Although not fully understood, the presence of multiplereceptors activated by a ligand, in addition to thepossibility of homo- and heterodimerization, undoubtedlyhas important implications for generating differentsignals and different outcomes. The specific ligand–

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receptor complex formed determines which tyrosineresidues are autophosphorylated, the cytoplasmicmediators that are engaged and hence which pathwaysare activated. Pathways activated include the Ras- andShc-activated MAPK pathway that is downstream of allErbB receptors, and the phosphatidyl inositol 3-kinasepathway that is directly or indirectly activated by mostErbB dimers [17,18]. Activation of intracellular signalingcascades culminates in events such as cell proliferation,differentiation, migration and survival, which are import-ant for pathological processes such as inflammation,tissue remodeling and wound healing. Some of theseprocesses bear striking similarities to the changes thatoccur in the ovarian follicle during ovulation.

EGF-like growth factor processing

The EGF-like growth factors are synthesized as trans-membrane precursors that can be released or ‘shed’ assoluble mature peptides by proteolytic cleavage of theectodomains at the membrane surface [20]. In particular,members of the ADAM family of proteases are thought tobe the major regulators of EGF-like growth factorshedding from the cell surface [21,22]. Tumor necrosisfactor a-converting enzyme (TACE/ADAM17) has beenimplicated in the shedding of amphiregulin, HB-EGF andTGF-a in several different cell lines [21–25], and inphorbol myristate acetate-induced shedding of epiregulinin mouse embryonic fibroblasts [21,23]. Consistent withthis view, TACE-deficient mice exhibit phenotypes similarto TGF-a-, HB-EGF- and EGFR-deficient mice [25–30]. Bycontrast, EGF shedding appears to be TACE independent[31], whereas ADAM10 but not TACE appears to be themajor convertase for betacellulin [21,23]. However, morethan one ADAMmight regulate proteolytic processing of aspecific EGFR ligand. For example, in addition to TACE,ADAM-9, -10 and -12 have been implicated in HB-EGFprocessing [13,24,32–34].

Although the unprocessed pro-forms of EGF-likegrowth factors have been shown to have biological activity[35–37], several lines of evidence suggest a crucial role forgrowth factor shedding in the activation of EGFRsignaling. For example, normal mouse development isdependent on soluble TGF-a rather than on the integralmembrane form, and requires functional TACE; therelease of soluble TGF-a was severely reduced inembryonic fibroblasts from TACE-deficient mice thatpresented with eye, hair and skin defects, abnormalitiesalso found in TGFa-deficient mice [25]. In HNSCC cells,cleavage of pro-amphiregulin at the cell surface to releasemature amphiregulin was required for GPCR-inducedEGFR activation and stimulation of cell proliferation andmigration [38].

Ligand-dependent EGFR transactivation by GPCRs

A model for the mechanism of GPCR-induced EGFRtransactivation has emerged and is referred to as the‘triple-membrane-passing-signaling’ pathway (Figure 1)[4,39]. In short, agonist binding to GPCRs leads tointracellular stimulation of specific metalloproteinases,cleavage and release of EGFR ligands at the cell surface,ligand binding and activation of the EGFR and


TRENDS in Endocrinology & Metabolism

RasRafMEK

?

PP

PPα

β γ

LigandPro-

ligand

GPCR(active)

MMP orADAMs

EGF-likefactor

Soluble

EGFR(active)

ERK/MAPK

Second-messengercascades

(a)

(b)

(c)

Figure 1. Model of EGFR transactivation. After GPCR stimulation, (a) undefined

intracellular signals activate specific transmembrane-bound metalloproteinases,

leading to (b) proteolytic cleavage of the ectodomain of EGF-like ligands at

the cell surface. The released or ‘shed’ growth factors activate the EGFR and

(c) subsequently the ERK/MAPK cascade. Because signals cross the membrane

three times, this pathway is referred to as the ‘triple-membrane-passing-signaling’

mechanism [4,39].


subsequent stimulation of the ERK/MAPK cascade. Thespecific metalloproteinase(s) involved in ligand sheddingis cell-type dependent. For example, in HNSCC cells,inhibition of TACE impaired the ability of LPA- orcarbachol-stimulated GPCRs to induce amphiregulinrelease and EGFR activation, demonstrating a functionalrole for TACE in mediating this event [38]. TACE is alsoimplicated in angiotensin II-stimulated HB-EGF shed-ding, EGFR activation and cell proliferation in ACHNkidney tumor cells [40]. However, in TccSup bladdercarcinoma cells, ADAM15 and not TACE mediatedLPA-induced EGFR transactivation that also involvedboth amphiregulin and TGF-a [40]. In other cells, matrixmetalloproteinase 2 (MMP2) or MMP9 might be respon-sible for the GPCR-mediated shedding [41].

Although it is clear that shedding is activated by GPCRoccupancy, the steps leading to metalloprotease activationare not well defined. In some cells, ligand shedding isdependent on PKC-induced activation of the metallo-protease [20,21]. In other instances, Src family kinasesappear to mediate EGFR pro-ligand cleavage [42], oralternatively might directly phosphorylate the EGFRand downstream adaptor proteins, such as Shc [6,43,44].Finally, in other systems the regulation of the synthesis ofthe EGF precursors might be a determining factor, as ingranulosa cells (see below).

GPCR–EGFR crosstalk in the neuroendocrine control of

reproduction

Studies of gonadotropin-releasing hormone (GnRH)-inducedsignals in hypothalamic- and pituitary-derived cellsindicate a role for MAPK and EGFR transactivation intransducing GPCR effects. GnRH is the master regulatorof the reproductive system, and pulsatile release of GnRHfrom hypothalamic neurons is crucial for regulating thesynthesis and release of the pituitary gonadotropins,follicle-stimulating hormone (FSH) and luteinizing


hormone (LH), and for exerting autocrine actions in theregulation of GnRH secretion [45]. FSH and LH, in turn,are essential for steroidogenesis, spermatogenesis, folli-culogenesis and ovulation.

Recently, Roelle et al. [41] demonstrated a requirementfor the gelatinases MMP2 and MMP9 in mediatingGnRH-induced EGFR transactivation and ERK/MAPKactivation in aT3-1 and LbT2 gonadotroph cells. These arethe first members of the MMP family reported toparticipate in this signaling mechanism. Roelle et al.[41] also showed that GnRH-induced EGFR transactiva-tion resulted in Fos and Jun expression in aT3-1 cells.Treatment of aT3-1 cells with the EGFR-specific inhibitorAG1478, the gelatinase inhibitor Ro28-2653 or the Srckinase inhibitor PP2 abrogated GnRH induction of c-Fosand c-Jun protein accumulation. Furthermore, ribozyme-targeted downregulation of either MMP2 or MMP9resulted in reduced c-Fos and c-Jun protein levels.

GnRH-induced synthesis of the a- and b-subunits ofFSH and LH in pituitary cells was also shown to involveMAPK activity. Treatment with PD098059, the specificinhibitor of MEK (MAPK activator), completely blockedGnRH-induced activation of the human a-subunit pro-moter in aT3-1 cells [46], and of ovine FSHb- and ratLHb-subunit promoters in LbT2 cells [47,48]. GnRH-regulated LHb-subunit protein accumulation alsooccurred via MAPK signaling in LbT2 cells [49]. WhetherGnRH-mediated MAPK activation and synthesis of thesefactors in the pituitary involves the EGFR is not yet clear.However, it is tempting to speculate that GnRH-inducedEGFR transactivation might be important during FSHand LH production.

Consistentwitha role forEGFRtransactivation inGnRHsignaling, GnRH induced metalloproteinase-dependentHB-EGF shedding in immortalized GT1-7 hypothalamicneuronal cells [50]. Interestingly, EGFR-induced signal-ing in hypothalamic astroglia, which are active partners ofneurons, is crucial for the production of glial-derivedmolecules that stimulate GnRH neurons to release GnRH[51]. Targeted disruption of EGFR signaling in astroglia ofadult female mice resulted in irregular estrous cycles anddecreased LH secretion, probably because of alterations inGnRH release, thus compromising reproductive function[52]. Taken together, these studies suggest a crucial rolefor interplay between the GPCR and EGFR pathways inthe neuroendocrine regulation of female reproduction.

GPCR–EGFR crosstalk in the regulation of ovulation

The importance of crosstalk between the GPCR and EGFRsignaling networks for reproductive processes is furthersupported by recent studies that implicate selectedmembers of the EGF-like growth factor family asmediators of LH action in the ovulatory follicle [53,54].In the ovary, the LH surge triggers a cascade of events inthe preovulatory follicle that are crucial for ovulation of afertilizable oocyte [55]. LH binds to its Gs-coupled receptoron theca and granulosa cells to activate adenylyl cyclase,increase intracellular cAMP levels and activate down-stream signaling pathways [56]. This leads to the spatialand temporal expression of specific genes that promoteoocyte meiotic resumption, cumulus expansion and



breakdown of the follicle wall – events necessary for therelease of a productive oocyte [55]. Because oocytes andcumulus cells express few or no LH receptors, LH effectson these cells are thought to be indirect [57]. Themechanism(s) by which LH signals are transduced tocumulus–oocyte complexes has not been fully elucidated,and is an area of great interest.

Recent studies show that amphiregulin, epiregulin andbetacellulin are rapidly and transiently induced by anovulatory dose of LH/human chorionic gonadotropin(hCG) in mouse and rat ovaries within 1–3 h of hormonestimulation (Figure 2) [53,54]. Furthermore, the LH surgeinduces a large increase inmRNA encoding epiregulin andamphiregulin in naturally cycling rats [58] and mice(M. Hsieh, unpublished). Transcripts for these growthfactors localized specifically to mural granulosa cells ofpreovulatory follicles, but not to the cumulus cells thatsurround the oocyte (with the exception of betacellulin)[53]. LH-induced synthesis of amphiregulin and epiregulinwas also detected in primary cultures of human granulosacells [59]. In addition, expression of the gene encodingepiregulin has emerged from several screenings of LH-regulated genes [60,61]. Park et al. [53] showed that thecorrespondingprotein is indeedproducedbygranulosa cells,as measured by immunological means and metaboliclabeling. Given that the presence of bioactive EGF-likegrowth factors in the follicular fluid has been previouslyreported [62], a substantial amount of data points to theLH-regulatedproduction ofEGF-like growth factors in vivo.

These EGF-like factors mimic many of the effects of LHin vitro. In a follicle culture model that recapitulates most


AC

cAMP

PKA

LHR

LH

Areg Ereg Btc

Gs

mRNA

DNA

EGF-likegenes

Figure 2. Model of LH regulation of EGF-like growth factor expression. The LH surge

or hCG stimulation causes a large increase inmRNA encoding amphiregulin (Areg),

epiregulin (Ereg) and betacellulin (Btc) via a cAMP-mediated process. Biochemical

and immunological evidence confirms the presence of these proteins on granulosa

cells. Abbreviations: AC, adenylyl cyclase; LHR, LH receptor.


of the events that occur in vivo, exogenously appliedamphiregulin and epiregulin induced oocyte meioticresumption to the same extent as did LH, but with afaster time course of induction [53]. Betacellulin waspartially effective in promoting this effect. These findingsare reminiscent of early studies showing that in vitrotreatment with EGF promotes oocyte maturation and is apotent stimulus of cumulus expansion [63–65]. Thus,epiregulin, amphiregulin and betacellulin are probablythe physiological ligands for the EGFR in the follicle. Thethree growth factors stimulated cumulus expansion inintact follicles, and the expression of the PTGS2 (prosta-glandinendoperoxidesynthase2, alsocalledcyclooxygenase2 or COX2), HAS2 (hyaluronan synthase 2), and TNFAIP6(tumor necrosis factor-a-induced protein 6, also calledTNF-stimulated gene 6 or TSG6) genes that are associatedwith this event [53,54]. In cultures of isolated cumulus–oocyte complexes where LH no longer has an effect, solubleamphiregulin, epiregulin or betacellulin were effective inpromoting cumulus expansion and oocyte maturation [53].Notably, these factors were unable to induce maturation ofdenuded oocytes, indicating a requirement for interactionsbetween theoocyteandcumuluscells [53].A similar absenceof direct effects on the isolated oocyte has been reportedwhen using EGF [64,66,67].

That LH effects are dependent on the activation of theEGF network is supported by the following observations.LH-induced maturation of cumulus–oocyte complexesrequires the activity of metalloproteases, because thepotent broad-spectrum metalloprotease inhibitor GM6001(also called Galardin or Ilomastat), but not a specificMMP2/MMP9 inhibitor, blocked LH-induced but notepiregulin-induced oocyte maturation in rat follicles [54].In addition, LH effects appear to involve activation of theEGFR found on granulosa and cumulus cells. LH/hCGinduced EGFR phosphorylation in mouse ovaries and incultured follicles; inhibition of EGFR activity by AG1478prevented LH-induced oocyte maturation and cumulusexpansion in vitro [53,54]. Moreover, injection of AG1478into rat ovarian bursas resulted in a significant decreasein LH-induced ovulation in the treated ovary, comparedwith the untreated controlateral ovaries and vehicle-treated ovaries [54]. An increased incidence of largefollicles containing entrapped oocytes, many of whichhad not undergone meiotic resumption and were sur-rounded by unexpanded cumulus cells, was observed intreated ovaries.

Based on the above findings, EGF-like growth factorsprobably function as mediators of LH action in theovulatory follicle. Because LH regulates mRNA andprotein synthesis, the transregulation of EGFR occurs ina time frame longer than the classic rapid regulation oflatent growth factor precursors. It remains to be deter-mined whether LH-stimulated shedding of pre-existinggrowth factor precursors has a role in the early stages ofgonadotropin signaling. A model of GPCR-mediatedEGFR transactivation can be presented, in which LHactivation of its specific GPCR leads to regulation ofEGF-like growth factors at the transcriptional level and atthe level of processing of ligands to their mature forms(Figure 3). The soluble EGF-like growth factors then act in



LHR

LH

?

PKA

AC

Granulosa cell

Gs

ERK/MAPKP P

P P P P

P P

cAMP

EGFRdimer

(Solubleligand)

Cumulus expansion

Cumulus cell

Oocyte meioticresumption

COX2TSG6HAS2

ERK/MAPK

AregEregBtc

MMP orADAMs

Pro-ligand

Figure 3. Model of EGFR transactivation in the regulation of LH-induced ovulation. LH activates the LHR on granulosa cells of preovulatory follicles, leading to stimulation of

cAMP-dependent signal cascades and the expression of mRNAs encoding the EGF-like growth factors amphiregulin (Areg), epiregulin (Ereg) and betacellulin (Btc). LH also

inducesmetalloproteinase-dependent processing of the pro-forms of these growth factors for the release ofmature peptides from the cell surface. The soluble growth factors

exert autocrine and paracrine effects through activation of the EGFR and ERK/MAPK signaling in granulosa cells and cumulus cells. This leads to oocyte meiotic resumption

and cumulus expansion, events crucial for the ovulatory process. Abbreviations: AC, adenylyl cyclase; LHR, LH receptor.


either an autocrine or paracrine manner to activate theEGFR, with subsequent activation of the MAPK cascade.Indeed, MAPK activity in cumulus cells has been shown tobe necessary for LH-induced cumulus expansion andoocyte maturation [68], and preliminary results suggestthat LH-induced MAPK activation in cumulus–oocytecomplexes is dependent on EGFR activity (M. Hsieh,unpublished).

Concluding remarks

The above studies show that EGFR transactivation is acommon mechanism by which an initial stimulus ispropagated to heterogeneous signal transduction path-ways. This transactivation was studied initially incultured cells in the realm of cancer biology or woundhealing. However, studies under more physiologicalconditions, particularly in the reproductive tract, supporta broader function for this signaling module. Thepossibility that activation of the EGF network plays animportant role in propagating the LH signal is an exampleof the involvement of this module in a physiologicalprocess. Understanding how this transactivation func-tions in the context of the follicle should provide a newperspective of this process. However, several major issuesstill need to be addressed. Most notably, the mechanism bywhich GPCRs activate metalloproteases involved inEGF-like growth factor shedding remains to be elucidated.Which metalloprotease(s) processes these growth factorsin the follicle is also unclear. Potential candidates can beinferred from what is known in other cell systems. This isan important issue because it might provide the specificityrequired for selective pharmacological manipulation.Finally, further studies are necessary to understand howEGFR transactivation in cumulus cells impacts on oocytemeiotic resumption. A better understanding of the role ofGPCR transactivation of the EGF network should open


new doors for the manipulation of physiological processes,including ovulation, or the improvement of in vitro cultureconditions of human oocytes.

AcknowledgementsThe work done in our laboratory was supported by National Institute ofChild Health and Human Development NIH grant HD20788 and by acooperative agreement (U54-HD31398) as part of the SpecializedCooperative Centers Program in Reproduction Research.

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Endea

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