Signaling by internalized G-protein-coupled receptorsDavide Calebiro1,2, Viacheslav O. Nikolaev1,2, Luca Persani3,4 and Martin J. Lohse1,2

1 Rudolf Virchow Center, DFG-Research Center for Experimental Biomedicine, University of Wurzburg, Wurzburg, Germany2 Institute of Pharmacology and Toxicology, University of Wurzburg, Wurzburg, Germany3 Dipartimento di Scienze Mediche, Universita degli Studi di Milano, Milan, Italy4 Laboratory of Experimental Endocrinology, Istituto Auxologico Italiano, Milan, Italy

Review

G-protein-coupled receptors (GPCRs) are cell surfacereceptors and are generally assumed to signal to secondmessengers such as cyclic AMP (cAMP) exclusively fromthe plasma membrane. However, recent studies indicatethat GPCRs can continue signaling to cAMP after intern-alization together with their agonists. Signaling frominside the cell is persistent and appears to triggerspecific downstream effects. Here, we will review theserecent data, which form the basis for a novel concept ofintracellular GPCR signaling and suggest new and intri-guing scenarios for the functions of GPCRs in the endo-cytic compartment. We propose that current models ofGPCR signaling should be revised to accommodate theability of receptors to change their signaling propertiesdepending on their subcellular localization.

IntroductionCells respond to environmental cues and communicatewith each other through the activation of receptors locatedon the cell surface. G-protein-coupled receptors (GPCRs)form the largest family of such receptors. They mediateeffects of neurotransmitters, hormones, ions, odorantsand light. Their signals are essentially mediated via theactivation of heterotrimeric G proteins and their effectors(e.g. adenylyl cyclase, phospholipase C, potassium andcalcium channels). Because of their involvement in a largenumber of physiological and pathological processes,GPCRs have been subject to intensive investigation andrepresentmajor targets for current pharmacological inter-vention [1].

Similar to other classes of receptors, prolonged stimu-lation of GPCRs often leads to their internalization intoendosomes, presumably via more than one internalizationpathway [1–3]. Although originally considered as a majormechanism of signal desensitization, the results of severalstudies performed over the past 15 years suggest otherfunctions for receptor internalization [4], most notably re-ceptor resensitization [1,5–7] and signaling to the mitogen-activated protein kinase (MAPK) cascade [8]. In addition,proteolytic fragments of internalized Frizzled GPCRs havebeen shown to translocate to the nucleus where they mightactivate gene transcription [9]. In spite of these obser-vations, it is generally believed that GPCR signalingto classical G-protein-dependent pathways, such as the

Corresponding authors: Calebiro, D. (davide.calebiro@toxi.uni-wuerzburg.de);Lohse, M.J. (lohse@toxi.uni-wuerzburg.de).

0165-6147/$ – see front matter � 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.

Gs-dependent activation of adenylyl cyclase, occurs exclu-sively at the cell surface. This view has been challenged bythree recent studies, which suggest that a previously unrec-ognized type of persistent GPCR signaling to cAMP canoccur after ligand–receptor internalization [10–12]. Here,wewill reviewtheevidence for the existenceofGPCR–cAMPsignaling pathways on endosomes, and their possible patho-physiological and pharmacological implications.

GPCR internalization and desensitizationThe molecular mechanisms of clathrin-dependent GPCRinternalization have been extensively investigated(reviewed in Refs. [1–3,13,14]). The trigger for receptorinternalization is the conformational change induced byagonist binding, which, apart from initiating G-protein-dependent signaling, transforms receptors into substratesof the G-protein-coupled receptor kinases (GRKs). As aresult, the ligand-occupied receptors become phosphory-lated at cytosolic Ser/Thr residues. Ligand-occupied andGRK-phosphorylated receptors rapidly recruit b-arrestins,an event that disrupts signaling to G proteins (see below).In addition, b-arrestins play a fundamental role in GPCRinternalization, as they promote clathrin-dependent endo-cytosis through interaction with elements of the endocy-totic machinery, such as the clathrin heavy chain or theclathrin adaptor protein AP2 [15,16]. Clathrin-coated pitsthen detach from the plasma membrane in a process thatrequires dynamin [17]. Once internalized, the ligand–re-ceptor complexes move along the endocytic pathway. Hereat least two possibilities exist. Either the receptors areseparated from their ligands and recycled back to the cellsurface or the receptors are transferred to the internalmembranes of late endosomes, an event that targets themto lysosomal degradation.

Desensitization of GPCRs occurs at different levels butis probably most relevant at the receptor level. Receptorinternalization was initially thought to play amajor role insignal desensitization, but then it became clear thatinternalization cannot make a large contribution to desen-sitization, as the latter occurs much faster than internal-ization, takes place already at the cell surface and isindependent of endocytosis [4]. A plethora of studies,particularly on the b2-adrenergic receptor, has clearlyshown that receptor phosphorylation by GRKs and thesubsequent binding of b-arrestins, along with proteinkinase A (PKA)- and protein kinase C (PKC)-dependentphosphorylation events at other sites on the receptor,

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Figure 1. Conventional model of GPCR signaling and trafficking. Binding of an agonist to a GPCR leads to the activation of heterotrimeric G proteins, which in turn stimulate

or inhibit effector proteins. The activation of downstream signaling cascades ultimately produces biological effects. In the case of persistent stimulation, GPCRs are

phosphorylated by GRKs and recruit b-arrestins (bArr), events responsible for fast signal desensitization. Subsequently, GPCRs are often internalized into endosomes.

Internalized GPCRs are either targeted to lysosomes for degradation or dephosphorylated and recycled back to the cell surface to sustain a new cycle of activation.

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constitute the main mechanisms of rapid GPCR desensi-tization [1,4].

Nevertheless, receptor endocytosis appears to haveother important functions in GPCR signaling. In the caseofGPCRs that recycle back to the cell surface, suchas theb2-adrenergic receptor, internalization seems to be involved inrestoring receptor responsiveness after desensitization.GPCR-containing endosomes have long been known to berich in phosphatases [18], and receptor internalization,followed by trafficking through this phophatase-containingcompartment and recycling to the cell surface, has beenshown to be required for receptor resensitization [1,5–7].

Internalized GPCRs that do not recycle are insteadrapidly targeted to lysosomes and degraded. This contrib-utes to the long-term process of receptor downregulation, amuch slower desensitization process, which requires hoursto days and consists of the reduction of the number ofreceptors present on the cell surface [1].

The conventional model of GPCR signaling and traffick-ing is depicted in Figure 1.

Lessons from receptor tyrosine kinase signalingEndosomes possess several characteristics that, at least inprinciple, make them ideal intracellular signaling plat-forms; among others, they have a high surface-to-volumeratio, which would favor ligand–receptor interactions, theyhave a unique lipid (high phosphatidylinositol 3-phosphate

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content) and protein composition permitting selectiverecruitment of signaling components, they move centripe-tally, thus potentially allowing the dissemination ofshort-range signals to compartments distant from the cellsurface (e.g. the nucleus) [3].

Early evidence that receptors can signal from endo-somes came from studies of receptor tyrosine kinases(RTKs) [19–21]. In these studies, it was shown that epi-dermal growth factor receptors (EGFRs), but also otherRTKs, can internalize together with their ligands andremain phosphorylated and active in endosomes. Inaddition, the principal components of the extracellularsignal-regulated kinase (ERK)–MAPK cascade have beenfound to be associated with RTKs on endosomes. Finally, ithas been shown that transfection with a dominant nega-tive mutant of dynamin or the RNAi-mediated depletion ofclathrin – treatments able to inhibit clathrin-dependentendocytosis – are associated with a blunted ERK phos-phorylation in response to RTK activation [22,23].

These findings provide good evidence that RTKs con-tinue to signal after internalization, but it has been hard toprove that this type of intracellular signaling producesspecific effects. Although several studies have suggestedthat intracellular RTK signaling is required for full ERKactivation [22–25], other studies have revealed contradic-tory conclusions [26–28]. Interestingly, at least in somecases, inhibiting RTK internalization and trafficking

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through the endocytic compartment has been shown tohave a negative impact on cell proliferation or growthfactor-induced cell motility [29]. Although it is difficultto provide an unequivocal interpretation of such contrast-ing results, it is possible that the contribution of endosomalsignaling can vary depending on the cell type and thespecific experimental conditions [3].

Perhaps the best example of a qualitative difference inRTK signaling from endosomes originates from the studyof nerve growth factor (NGF) signaling. In neurons, NGFsignaling activates the TrkA receptor in distal axon ter-mini to promote cell survival. This signal must be trans-ferred from the distal axon termini to the nucleus to inducethe transcription of antiapoptotic genes. Interestingly, ithas been shown that a retrograde transport of endosomescontaining active TrkA signaling complexes is required forERK5 activation and phosphorylation of the cAMP respon-sive element-binding protein (CREB) in the nucleus. Incontrast, activation of TrkA on the cell surface results in alocal activation of ERK1 and ERK2, which is insufficientfor the phosphorylation of CREB and the induction of thesurvival program [30].

‘Non-classical’ GPCR signaling at intracellularmembranesIn analogy with the findings on RTKs, GPCR signaling toMAPKs has also been proposed to occur at endosomes. Thefirst evidence came from experiments where a dynamindominant negative mutant was shown to inhibit ERKactivation stimulated by the b2-adrenergic receptor [8].Subsequently, it was shown that certain GPCRs remainassociated with b-arrestins in endosomes [31] and that b-arrestins can bind to several components of the MAPKpathways [32,33]. In light of these findings, it has beenproposed that b-arrestins function as membrane-tetheredscaffolds capable of recruiting elements of the MAPK path-ways to membranes of internalizing vesicles or endosomes,thus facilitating ERK activation (Figure 2a). Furthermore,by anchoring activated ERK to endosomes, b-arrestinsmight prevent ERK translocation to the nucleus, thusfavoring cytoplasmic ERK signaling [13,31,33,34]. How-ever, similar to RTKs, the b-arrestin-dependent activationof ERK apparently also occurs on the plasma membrane,and the exact physiological role of the intracellular acti-vation of ERK is still debated. Furthermore, this type ofERK activation is only one of many pathways wherebyGPCRs can activate MAPK signaling [35].

In contrast to the b-arrestin-dependent activation ofMAPKs, G-protein-dependent signaling is generallyassumed to be restricted to the plasma membrane, andinternalization is thought to disrupt receptor–G proteinsignaling. However, this view has been challenged byrecent studies. In a study performed in the budding yeastSaccharomyces cerevisiae, Slessareva et al. have shownthat the GPCR Ste2 can activate the phosphatidylinositol3-kinase Vsp34 at endosomes [36,37] (Figure 2b). Stimu-lation of Ste2 by the pheromone a-factor results in theactivation of heterotrimeric G proteins with release ofGpa1, the yeast homolog of mammalian Ga, from theGbg complex. Gpa1 then translocates to endosomes, whereit is thought to activate Vsp34, and consequently the

production of phosphatidylinositol 3-phosphate (PI3P).This local increase in PI3P would then trigger the recruit-ment of FYVE-containing signaling proteins, thus result-ing in an enhanced activation of MAPKs and Cdc42. Thiswas the first direct demonstration that G-protein-depend-ent signaling can also occur on intracellular membranes.

In addition, there are other reports suggesting that ‘non-classical’ G-protein-dependent signaling pathways mightexist at intracellular compartments. Heterotrimeric Gproteins are frequently found on the membranes of intra-cellular organelles such as secretory granules, endosomes,the endoplasmic reticulum (ER), the Golgi complex and thetrans-Golgi network (TGN), where they are thought to playroles in vesicle trafficking [36,38,39]. Indeed, several linesof evidence indicate that G proteins are assembled andincorporated into membranes in the ER and in the Golgicomplex, and are able to affect both Golgi structural organ-ization and transport activities. In the TGN, Gs appears tohave positive effects on vesicle fission, whereas Gi/o canhave negative effects. A similar situation has beenreported for endocytosis and exocytosis, where differenttypes of G proteins seem to have different effects on vesiclefusion. Furthermore, Gbg subunits also appear to play arole, either direct or indirect, in vesicle trafficking.Although the mechanism of activation of heterotrimericG proteins on these intracellular compartments is largelyunknown, an alternative pathway (i.e. GPCR-indepen-dent) has generally been advocated. Interestingly, Gar-cia-Regalado et al. have recently shown that Gbg

interacts with Rab11a and that, after activation of lysopho-sphatidic acid (LPA) receptors, the resulting complex islocalized in early and recycling endosomes [40]. Theauthors suggest that this interaction would promote therecruitment of PI3Kg and the phosphorylation of Akt onendosomes, and that the activation of this pathway mightcontribute to the proliferative and antiapoptotic effects ofLPA (Figure 2c). In another study, Diaz Anel has suggestedthat, in response to an as yet unidentified GPCR, Gbg cantranslocate to membranes of the TGN to activate phospho-lipase Cb3 with the formation of diacylglycerol [41]. Thiswould trigger the activation of protein kinase Ch andsubsequently protein kinaseD, finally leading to the fissionof cargo-filled vesicles from the TGN (Figure 2d). Althoughmost evidence provided is indirect, and although it is notclear whether the involved GPCRs stay at the cell surfaceor are targeted together with G proteins to the proposedintracellular signaling compartments, these two publi-cations suggest the existence of a link between GPCRsignaling and the activation of G proteins at intracellularmembranes.

Persistent GPCR signaling to cAMP at endosomesVery recently, the emerging concept of ‘non-classical’ endo-somal GPCR signaling has been complemented by findingsalso suggesting classical, G-protein-dependent signaling ofintracellular GPCRs. Data from three groups providestrong evidence for persistent signaling to adenylyl cyclaseby internalized GPCRs.

To monitor GPCR–cAMP signaling directly in livingcells, we have recently developed a transgenic mouse[10] with ubiquitous expression of a fluorescent reporter

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Figure 2. ‘Non-classical’ GPCR signaling at intracellular membranes. (a) After internalization in complex with b-arrestin, some GPCRs (i.e. the b2-adrenergic receptor) can

activate the MAPK cascade on endosomes via a b-arrestin-dependent and G-protein-independent mechanism. (b) In yeast, stimulation of the Ste2 receptor with the

pheromone a-factor leads to release of the Ga subunit (Gpa1) from the Gbg complex and its translocation to endosomes. Here, Gpa1 activates the phosphatidylinositol 3-

kinase Vsp34. The resulting increase of phosphatidylinositol 3-phosphate (PI3P) on endosomal membranes ultimately leads to the activation of MAPK and Cdc42 pathways.

(c) LPA receptors activate PI3Kg via Gbg, thus leading to phosphatidylinositol 3,4,5-trisphosphate (PIP3) production and Akt activation on the plasma membrane. Thereafter,

Gbg translocates to early and recycling endosomes and interacts with Rab11a. Internalized Gbg can continue to activate PI3Kg and Akt on endosomes. (d) In response to an

as yet unidentified GPCR, Gbg might translocate to membranes of the TGN and activate phospholipase Cb3 (PLCb3). The resulting increase of DAG stimulates protein kinase

Ch (PKCh) and protein kinase D (PKD), leading to the fission of cargo-filled vesicles from the TGN.

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for cAMP [42]. The thyroid stimulating hormone (TSH),secreted by the anterior pituitary, binds to the TSH re-ceptor (TSHR) located on the basolateral membrane ofthyroid cells. At physiological TSH concentrations, theTSHR is predominantly coupled to Gs, and therefore itseffects are largely mediated by cAMP. Earlier studies,mostly performed on transfected cells, have shown that

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after prolonged TSH stimulation, the TSHR is internalizedand then recycled to the cell surface [43,44]. We haveutilized thyroid follicles isolated from the cAMP reportermouse and fluorescent TSH to study the spatiotemporaldynamics of TSH signaling. In primary thyroid cells stimu-lated with TSH, the receptor and its ligand are rapidlyand efficiently internalized into a perinuclear vesicular

Figure 3. Differential outcomes of GPCR signaling from the plasma membrane and

intracellular compartments. A FRET reporter for cAMP (Epac1–camps) [42] is used

to monitor intracellular cAMP levels. (a) TSHR signaling to cAMP from the plasma

membrane is reversible. (b) Upon prolonged stimulation with TSH, both receptor

and ligand are cointernalized into a perinuclear tubulovescicular structure that also

contains Gs and adenylyl cyclase. A representative image of fluorescent TSH

(green) and Gs (red), where yellow color is indicative of colocalization, is shown.

Here, Gs is detected by immunofluorescence with a specific antibody. (c) Signaling

from internalized TSHR is persistent (i.e. it continues after removal of TSH) and is

more efficient in stimulating VASP phosphorylation and actin depolymerization.

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compartment; however, overall intracellular cAMP levelsremain high. Several findings, including the effects ofendocytosis inhibitors and the results of cell fractionationexperiments, provide strong evidence that heterotrimericG proteins and adenylyl cyclases are also present onendosomes and that internalized TSH–TSHR complexescontinue to stimulate cAMP production. Whereas TSHR–

cAMP signaling from the plasma membrane is rapidlyreversible, signaling from internalized receptors continuesafter removal of TSH. Furthermore, TSHR signaling fromendosomes might be required for efficient thyroglobulinendocytosis and thus thyroid hormone release, assuggested by the fact that TSHR internalization is neededto induce full phosphoryation of the vasodilator-stimulatedphosphoprotein (VASP), a key regulator of actin dynamics,and cause actin depolymerization. Taken together, thesefindings demonstrate for the first time not only that GPCRsignaling to cAMP can continue after internalization butalso that GPCR signaling from endosomes can lead to bothquantitative and qualitative differences in signaling out-comes [10] (Figure 3).

Soon after the publication of this report, Ferrandon et al.published the results of a study on the parathyroid hor-mone (PTH) receptor (PTHR), which suggest a similar typeof intracellular cAMP signaling [11]. PTHR has two dis-tinct physiological ligands: PTH, a circulating hormone,and PTH-related peptide (PTHrP), a paracrine factor; thetwo ligands trigger cAMP responses of different durations.In their study, Ferrandon et al. utilized various fluor-escence resonance energy transfer (FRET) reporters, in-cluding the one for cAMP, and fluorescently labeled ligandsto compare the kinetics of PTH and PTHrP signaling. Theyfound that PTH stimulation induces a rapid internaliz-ation of ligand–receptor complexes into early endosomes inassociation with adenylyl cyclase. Similarly to what wasobserved in the case of the TSHR, internalization of PTHR–

PTH complexes was not associated with desensitization ofthe cAMP response but rather with persistent signaling. Incontrast, PTHrP actions were completely reversible andappeared restricted to the cell surface. In spite of somepossible caveats, including the use of a non-physiologicalmodel (HEK cells overexpressing the PTHR) and the lackof direct proof to support PTHR signaling to cAMP onendosomes, these results provide further evidence forthe existence of a previously unrecognized pathway linkingGPCRs to adenylyl cyclase activation on endosomes.

Although the two above-mentioned studies stronglysupport the existence of Gs-dependent signaling on endo-somes, yet another very recent paper suggests that Gi-dependent signaling might also be occurring intracellu-larly. The sphingosine-1-phosphate receptor 1 (S1P1), a Gi/Gq-coupled receptor, is the main target of the immunomo-dulator drug FTY720, which is used in the treatment ofmultiple sclerosis [45]. Interestingly, Mullershausen et al.have found that activated S1P1 receptors continue tosignal to Gi for hours, as shown by a persistent inhibitionof forskolin-stimulated cAMP production, in spite of con-sistent internalization [12]. Moreover, analogs with lowerhydrophobicity but conserved potency and efficacy wereunable to promote persistent signaling. These findingssupport the view that S1P1 receptors can continue to signalto Gi and thus lead to persistent adenylyl cyclase inhibitionat intracellular sites. In contrast, the Gq-dependent acti-vation of the PLC–Ca2+ signaling pathway appearsrestricted to the cell surface.

Functional consequences of GPCR–cAMP signaling onendosomesAlthough these new findings clearly support the existenceof GPCR–cAMP signaling pathways on endosomes, ourunderstanding of their functional relevance is still limited.What appears clear is that, differently from what is occur-ring at the cell surface, GPCR–cAMP signaling on endo-somes is persistent. This phenomenon might beparticularly relevant in vivo, where the access to ligandsis often limited and can vary over time. This is the case forseveral hormones, including TSH [46], that are secretedwith a circadian rhythm or for neurotransmitters, whosepulsatile secretion results in submillisecond transients.Thus, as anticipated by Miaczynska et al. [19], sustainedcAMPproduction from internalized receptors can provide a‘memory’ mechanism, allowing cells to react with pro-longed responses to short-term stimuli. As suggested bythe case of the PTHR, this type of intracellular signalingmight occur for certain (e.g. PTH) but not for other (e.g.PTHrP) ligands and thus could explain differences be-tween their durations of action.

In addition, cAMP signaling from endosomes mighthave different outcomes than signaling from the plasmamembrane. In the case of the TSHR, signaling from insidethe cell appears to be more efficiently coupled to the PKA-dependent reorganization of actin cytoskeleton, an eventthat is involved in thyroid hormone production and release.This could be explained if the receptors need to be trans-ferred into the interior of the cell to activate PKA effi-ciently. However, such a localized pattern of cAMPproduction makes sense only if cAMP cannot freely diffuse

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Figure 4. Persistent GPCR signaling to cAMP at endosomes. (a) The TSHR and the PTHR are coupled to Gs and stimulate cAMP production at the plasma membrane. After

internalization together with their ligands into endosomes containing both Gs and adenylyl cyclase, they continue to stimulate adenylyl cyclase, leading to persistent cAMP

signaling from the interior of the cell. (b) Binding of phosphorylated FTY720 (FTY720P), an immunomodulator drug, to the S1P1 receptor activates Gi and inhibits adenylyl

cyclase activity at the plasma membrane. The S1P1 receptor continues to inhibit cAMP production after internalization. GPCR signaling to cAMP at endosomes can lead to

specific signaling outcomes.

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inside cells. Indeed, although restricted cAMP diffusionhas been advocated to account for the specificity of certainGPCR effects, whether or not cAMP appears to freelydiffuse seems to depend both on the cell type and on theexperimental set-up. Thus, restricted [47,48] as well as free[42] diffusion has been observed in primary neurons,whereas in cardiac myocytes both cAMP-diffusion [49,50]and cAMP-dependent signaling [51,52] appear to bespatially restricted. Furthermore, the existence of cAMPgradients is predicted on the basis of the spatial segre-gation of adenylyl cyclases on the membrane and certainphosphodiesterase isoforms in the cytosol [53]. In thisregard, GPCR–cAMP signaling from intracellular sitesmight provide a new basis to explain the activation oftargets located at distant sites, such as the nucleus, inthe presence of restricted cAMP diffusion.

Another example of how signaling from inside the cellcan differ from that occurring at the cell surface comes fromthe above-mentioned study on S1P1 receptors. S1P1 recep-tors are coupled to both Gi and Gq. Thus, stimulation withFTY720–phosphate results in both inhibition of cAMPproduction and increase of intracellular Ca2+ concen-trations. However, although signaling from the plasmamembrane seems to be coupled to both pathways, signaling

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from inside the cell appears to just inhibit cAMP pro-duction, without having any effects on Ca2+ levels. Accord-ingly, receptor internalization, coupled to the selectivelocalization of effectors on different cellular membranes(e.g. plasma membrane or endosomes), could provide thebasis for a temporal regulation of receptor coupling todifferent downstream signaling pathways.

Concluding remarksBased on these recent results we propose a new model ofGPCR signaling (Figure 4). Several GPCRs are interna-lized together with their ligands (and perhaps with Gproteins and adenylyl cyclases) in endosomes or otherintracellular compartments. Here, at least some of them,namely TSHR, PTHR and S1P1 receptors, find themachin-ery required for cAMP production. As both ligands andreceptors remain confined in endosomes for some time, thismechanism permits prolonged signaling even afterremoval of the agonist from the extracellular space. Inaddition, as suggested by the effects of internalized TSHRon actin cytoskeleton, this new type of signaling can pro-duce specific functional outcomes.

The advent of genetically encoded fluorescent reportersfor monitoring cAMP levels in living cells [42,54–56] has

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led to new insights into themechanisms of GPCR signalingand signal compartmentalization. Notably, the use of afluorescent cAMP reporter was instrumental for both find-ings on TSHR and PTHR persistent signaling after intern-alization. In addition, the availability of a transgenicmouse with ubiquitous expression of a fluorescent cAMPreporter allowed us to study TSHR signaling directly innative thyroid follicles (i.e. very close to physiologicalconditions).We believe that this and other types of imagingapproaches will play an important role in further investi-gations of the fate and functions of GPCRs after internal-ization.

Nevertheless, several technical limitations exist thatmight hinder our progress. First, the currently usedmethods for inhibiting GPCR endocytosis (which wereapplied with some success to study short-term responses)appear inadequate to analyze long-term effects (such asthose on gene transcription), because they targetmoleculessuch as clathrin or dynamin, which are implicated not onlyin endocytosis but also in other cellular processes. Second,the proteins involved in the initial steps of GPCR signaling(i.e. receptors, G proteins and effectors) are generallyexpressed at very low levels in native cells, which limitsour capabilities of fully appreciating their subcellularlocalization and interactions in physiological conditions.Third, the endocytic compartment is highly intricate anddynamic, which adds another level of complexity. There-fore, further improvements in our capabilities of monitor-ing signaling events in living cells as well as more selectivestrategies to inhibit GPCR endocytosis will most likely berequired to further advance our knowledge in this field.

Another issue to consider is that endosomes representonly one of several possible sites of intracellular GPCRsignaling. For instance, some GPCRs, such as the chemo-kine receptor CCR5 or the gonadotropin-releasing hormonereceptor, appear to be largely retained in the ER and Golgicomplex, even when observed in their natural context[57,58]. In addition, there is evidence for early associationof GPCRs, G proteins and their effectors in these intracellu-lar compartments [59]. As suggested by a recent study onintracellularly retained vasopressin V2 receptor mutants,such biosynthetic compartments might contain functionalGPCRs that can be activated by cell-permeable agonists[60]. Thus, particular care should be taken when drawingconclusions on the subcellular localization of GPCR-initiated signals in response to lipophilic ligands.

With this inmind, there are several important questionsthat can and need to be answered. First, is persistent GPCRsignaling to cAMP limited to a few receptors or is it a moregeneral phenomenon? And if it is a peculiar feature ofcertain receptors only, what are the determinants? Indeed,even if persistent cAMP signaling after internalizationmight be common to other GPCRs, its contribution to theoverall signaling outcome might vary as a consequence ofcomplex interactions between factors suchas ligandaffinity,degree of receptor phosphorylation, affinity for b-arrestinsand the fate of both receptor and ligand after internaliz-ation, not to mention cell-specific differences in the compo-sition and/or organization of signaling complexes. Based onthese considerations, it might not be by chance that thisphenomenon has been described for TSHR and PTHR, two

peptide/protein hormone receptors that form rather stablecomplexeswith their ligands. Second, can signaling throughother G proteins also occur at intracellular membranes?Third, what is the physiological and pathophysiologicalrelevance of GPCR signaling at endosomes? For instance,persistentTSHRsignalingatendosomesmightplayarole inthepathogenesis ofGraves’ disease or indisorders causedbyTSHR activating mutations. Fourth, what are its pharma-cological implications? As already shown for S1P1 and PTHreceptors, different ligands can preferentially induceplasma membrane or intracellular signaling, which canbe relevant for future drug design. Furthermore, interferingwith endocytosis might become a new tool for fine tuningGPCR signaling and therefore a new strategy for thera-peutic intervention.

Although further studies will be required to fullyappreciate the relevance of GPCR signaling after intern-alization, endosomes should no longer be viewed as ‘sinks’for receptors but rather as dynamic signaling platforms,whose intriguing functions in GPCR signaling remain to beexplored.

AcknowledgementResearch by the authors referred to in this publication is supported bygrants from the European Research Council (Advanced Grant TOPAS)and the Deutsche Forschungsgemeinschaft (SFB487).

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