Signaling by Internalized

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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 Wu rzburg, Wu rzburg, Germany2 Institute of Pharmacology and Toxicology, University of Wu rzburg, Wu rzburg, Germany3 Dipartimento di Scienze Mediche, Universita degli Studi di Milano, Milan, Italy4 Laboratory of Experimental Endocrinology, Istituto Auxologico Italiano, Milan, Italy

G-protein-coupled receptors (GPCRs) are cell surface

receptors and are generally assumed to signal to second

messengers such as cyclic AMP (cAMP) exclusively from

the plasma membrane. However, recent studies indicate

that GPCRs can continue signaling to cAMP after intern-

alization together with their agonists. Signaling from

inside the cell is persistent and appears to trigger

specific downstream effects. Here, we will review these

recent data, which form the basis for a novel concept of

intracellular GPCR signaling and suggest new and intri-

guing scenarios for the functions of GPCRs in the endo-

cytic compartment. We propose that current models of

GPCR signaling should be revised to accommodate the

ability of receptors to change their signaling properties

depending on their subcellular localization.

Introduction

Cells respond to environmental cues and communicate

with each otherthrough the activation of receptors located

on the cell surface. G-protein-coupled receptors (GPCRs)

form the largest family of such receptors. They mediateeffects of neurotransmitters, hormones, ions, odorants

and light. Their signals are essentially mediated via the

activation of heterotrimeric G proteins and their effectors

(e.g. adenylyl cyclase, phospholipase C, potassium and

calcium channels). Because of their involvement in a large

number of physiological and pathological processes,

GPCRs have been subject to intensive investigation and

representmajor targets for current pharmacological inter-

vention [1].

Similar to other classes of receptors, prolonged stimu-

lation of GPCRs often leads to their internalization into

endosomes, presumably via more than one internalization

pathway[13]. Although originally considered as a major

mechanism of signal desensitization, the results of several

studies performed over the past 15 years suggest other

functions for receptor internalization [4], most notably re-

ceptor resensitization[1,57]and signaling to the mitogen-

activated protein kinase (MAPK) cascade [8]. In addition,

proteolytic fragments of internalized Frizzled GPCRs have

been shown to translocate to the nucleus where they might

activate gene transcription [9]. In spite of these obser-

vations, it is generally believed that GPCR signaling

to classical G-protein-dependent pathways, such as the

Gs-dependent activation of adenylyl cyclase, occurs exclu-

sively at the cell surface. This view has been challenged by

three recent studies, which suggest that a previously unrec-

ognized type of persistent GPCR signaling to cAMP can

occur after ligandreceptor internalization [1012]. Here,

we will reviewthe evidencefor theexistence ofGPCRcAMP

signaling pathways on endosomes, and theirpossible patho-

physiological and pharmacological implications.

GPCR internalization and desensitization

The molecular mechanisms of clathrin-dependent GPCR

internalization have been extensively investigated

(reviewed in Refs. [13,13,14]). The trigger for receptor

internalization is the conformational change induced by

agonist binding, which, apart from initiating G-protein-

dependent signaling, transforms receptors into substrates

of the G-protein-coupled receptor kinases (GRKs). As a

result, the ligand-occupied receptors become phosphory-

lated at cytosolic Ser/Thr residues. Ligand-occupied and

GRK-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 GPCR

internalization, as they promote clathrin-dependent endo-

cytosis through interaction with elements of the endocy-

totic machinery, such as the clathrin heavy chain or the

clathrin adaptor protein AP2[15,16]. Clathrin-coated pits

then detach from the plasma membrane in a process that

requires dynamin [17]. Once internalized, the ligandre-

ceptor complexes move along the endocytic pathway. Here

at least two possibilities exist. Either the receptors are

separated from their ligands and recycled back to the cell

surface or the receptors are transferred to the internal

membranes of late endosomes, an event that targets them

to lysosomal degradation.Desensitization of GPCRs occurs at different levels but

is probably most relevant at the receptor level. Receptor

internalization was initially thought to play a major role in

signal desensitization, but then it became clear that

internalization 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 is

independent of endocytosis [4]. A plethora of studies,

particularly on the b2-adrenergic receptor, has clearly

shown that receptor phosphorylation by GRKs and the

subsequent binding of b-arrestins, along with protein

kinase A (PKA)- and protein kinase C (PKC)-dependent

phosphorylation events at other sites on the receptor,

Review

Corresponding authors: Calebiro, D. ([email protected]);

Lohse, M.J. ([email protected]).

0165-6147/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.02.002 Available online 18 March 2010 221
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constitute the main mechanisms of rapid GPCR desensi-

tization[1,4].

Nevertheless, receptor endocytosis appears to have

other important functions in GPCR signaling. In the case

of GPCRs that recycle back to thecellsurface,suchas theb2-

adrenergic receptor, internalization seems to be involved in

restoring receptor responsiveness after desensitization.

GPCR-containing endosomes have long been known to be

rich in phosphatases [18], and receptor internalization,

followed by trafficking through this phophatase-containing

compartment and recycling to the cell surface, has been

shown to be required for receptor resensitization[1,57].

Internalized GPCRs that do not recycle are instead

rapidly targeted to lysosomes and degraded. This contrib-

utes to the long-term process of receptor downregulation, amuch slower desensitization process, which requires hours

to days and consists of the reduction of the number of

receptors 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 signaling

Endosomes possess several characteristics that, at least in

principle, make them ideal intracellular signaling plat-

forms; among others, they have a high surface-to-volume

ratio, which would favor ligandreceptor interactions, they

have a unique lipid (high phosphatidylinositol 3-phosphate

content) and protein composition permitting selective

recruitment of signaling components, they move centripe-

tally, thus potentially allowing the dissemination of

short-range signals to compartments distant from the cell

surface (e.g. the nucleus)[3].

Early evidence that receptors can signal from endo-

somes came from studies of receptor tyrosine kinases

(RTKs)[1921]. In these studies, it was shown that epi-

dermal growth factor receptors (EGFRs), but also other

RTKs, can internalize together with their ligands and

remain phosphorylated and active in endosomes. In

addition, the principal components of the extracellular

signal-regulated kinase (ERK)MAPK cascade have been

found to be associated with RTKs on endosomes. Finally, it

has been shown that transfection with a dominant nega-tive mutant of dynamin or the RNAi-mediated depletion of

clathrin treatments able to inhibit clathrin-dependent

endocytosis 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 to

prove that this type of intracellular signaling produces

specific effects. Although several studies have suggested

that intracellular RTK signaling is required for full ERK

activation[2225], other studies have revealed contradic-

tory conclusions [2628]. Interestingly, at least in some

cases, inhibiting RTK internalization and trafficking

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.

Review Trends in Pharmacological Sciences Vol.31 No.5

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through the endocytic compartment has been shown to

have a negative impact on cell proliferation or growth

factor-induced cell motility [29]. Although it is difficult

to provide an unequivocal interpretation of such contrast-

ing results, it is possible that the contribution of endosomal

signaling can vary depending on the cell type and the

specific experimental conditions[3].

Perhaps the best example of a qualitative difference in

RTK signaling from endosomes originates from the studyof nerve growth factor (NGF) signaling. In neurons, NGF

signaling 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 induce

the transcription of antiapoptotic genes. Interestingly, it

has been shown that a retrograde transport of endosomes

containing active TrkA signaling complexes is required for

ERK5 activation and phosphorylation of the cAMP respon-

sive element-binding protein (CREB) in the nucleus. In

contrast, activation of TrkA on the cell surface results in a

local activation of ERK1 and ERK2, which is insufficient

for the phosphorylation of CREB and the induction of the

survival program[30].

Non-classical GPCR signaling at intracellular

membranes

In analogy with the findings on RTKs, GPCR signaling to

MAPKs has also been proposed to occur at endosomes. The

first evidence came from experiments where a dynamin

dominant negative mutant was shown to inhibit ERK

activation stimulated by the b2-adrenergic receptor [8].

Subsequently, it was shown that certain GPCRs remain

associated with b-arrestins in endosomes[31]and that b-

arrestins can bind to several components of the MAPK

pathways[32,33]. In light of these findings, it has been

proposed that b-arrestins function as membrane-tethered

scaffolds 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-arrestins

might prevent ERK translocation to the nucleus, thus

favoring cytoplasmic ERK signaling[13,31,33,34]. How-

ever, similar to RTKs, the b-arrestin-dependent activation

of 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 of

ERK activation is only one of many pathways whereby

GPCRs can activate MAPK signaling[35].

In contrast to the b-arrestin-dependent activation of

MAPKs, G-protein-dependent signaling is generallyassumed to be restricted to the plasma membrane, and

internalization is thought to disrupt receptorG protein

signaling. However, this view has been challenged by

recent studies. In a study performed in the budding yeast

Saccharomyces cerevisiae, Slessareva et al. have shown

that the GPCR Ste2 can activate the phosphatidylinositol

3-kinase Vsp34 at endosomes[36,37] (Figure 2b). Stimu-

lation of Ste2 by the pheromone a-factor results in the

activation of heterotrimeric G proteins with release of

Gpa1, the yeast homolog of mammalian Ga, from the

Gbg complex. Gpa1 then translocates to endosomes, where

it 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. This

was the first direct demonstration that G-protein-depend-

ent signaling can also occur on intracellular membranes.

In addition, there areother reports suggesting that non-

classical G-protein-dependent signaling pathways might

exist 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 the

trans-Golgi network (TGN), where they are thought to play

roles in vesicle trafficking[36,38,39]. Indeed, several lines

of evidence indicate that G proteins are assembled and

incorporated into membranes in the ER and in the Golgi

complex, and are able to affect both Golgi structural organ-

ization and transport activities. In the TGN, Gsappears to

have positive effects on vesicle fission, whereas Gi/o can

have negative effects. A similar situation has been

reported for endocytosis and exocytosis, where different

types of G proteins seem to have different effects on vesiclefusion. Furthermore, Gbg subunits also appear to play a

role, either direct or indirect, in vesicle trafficking.

Although the mechanism of activation of heterotrimeric

G proteins on these intracellular compartments is largely

unknown, 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 is

localized in early and recycling endosomes [40]. The

authors suggest that this interaction would promote the

recruitment of PI3Kg and the phosphorylation of Akt on

endosomes, and that the activation of this pathway might

contribute to the proliferative and antiapoptotic effects of

LPA (Figure 2c). In another study, Diaz Anel has suggested

that, in response to an as yet unidentified GPCR, Gbgcan

translocate to membranes of the TGN to activate phospho-

lipase Cb3 with the formation of diacylglycerol [41]. This

would trigger the activation of protein kinase Ch and

subsequently protein kinase D, finally leading to the fission

of cargo-filled vesicles from the TGN (Figure 2d). Although

most evidence provided is indirect, and although it is not

clear whether the involved GPCRs stay at the cell surface

or are targeted together with G proteins to the proposed

intracellular signaling compartments, these two publi-

cations suggest the existence of a link between GPCR

signaling and the activation of G proteins at intracellularmembranes.

Persistent GPCR signaling to cAMP at endosomes

Very recently, the emerging concept of non-classical endo-

somal GPCR signaling has been complemented by findings

also suggesting classical, G-protein-dependent signaling of

intracellular GPCRs. Data from three groups provide

strong evidence for persistent signaling to adenylyl cyclase

by internalized GPCRs.

To monitor GPCRcAMP signaling directly in living

cells, we have recently developed a transgenic mouse

[10]with ubiquitous expression of a fluorescent reporter


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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 of

thyroid cells. At physiological TSH concentrations, the

TSHR is predominantly coupled to Gs, and therefore its

effects are largely mediated by cAMP. Earlier studies,

mostly performed on transfected cells, have shown that

after prolonged TSH stimulation, the TSHR is internalized

and then recycled to the cell surface [43,44]. We have

utilized thyroid follicles isolated from the cAMP reporter

mouse and fluorescent TSH to study the spatiotemporal

dynamics of TSH signaling. In primary thyroid cells stimu-

lated with TSH, the receptor and its ligand are rapidly

and efficiently internalized into a perinuclear vesicular

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 Gasubunit (Gpa1) from the Gbgcomplex 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,

Gbgtranslocates to early and recycling endosomes and interacts with Rab11a. Internalized Gbgcan continue to activate PI3Kgand 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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compartment; however, overall intracellular cAMP levels

remain high. Several findings, including the effects of

endocytosis inhibitors and the results of cell fractionation

experiments, provide strong evidence that heterotrimeric

G proteins and adenylyl cyclases are also present on

endosomes and that internalized TSHTSHR complexes

continue to stimulate cAMP production. Whereas TSHR

cAMP signaling from the plasma membrane is rapidly

reversible, signaling from internalized receptors continues

after removal of TSH. Furthermore, TSHR signaling from

endosomes might be required for efficient thyroglobulin

endocytosis and thus thyroid hormone release, as

suggested by the fact that TSHR internalization is needed

to induce full phosphoryation of the vasodilator-stimulated

phosphoprotein (VASP), a key regulator of actin dynamics,

and cause actin depolymerization. Taken together, these

findings demonstrate for the first time not only that GPCR

signaling to cAMP can continue after internalization but

also that GPCR signaling from endosomes can lead to both

quantitative and qualitative differences in signaling out-

comes[10](Figure 3).

Soon after the publication of this report, Ferrandonet 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; the

two 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 ligands

to compare the kinetics of PTH and PTHrP signaling. They

found that PTH stimulation induces a rapid internaliz-

ation of ligandreceptor complexes into early endosomes in

association with adenylyl cyclase. Similarly to what was

observed in the case of the TSHR, internalization of PTHR

PTH complexes was not associated with desensitization of

the cAMP response but rather with persistent signaling. In

contrast, PTHrP actions were completely reversible and

appeared restricted to the cell surface. In spite of some

possible caveats, including the use of a non-physiological

model (HEK cells overexpressing the PTHR) and the lack

of direct proof to support PTHR signaling to cAMP on

endosomes, these results provide further evidence for

the existence of a previously unrecognized pathway linkingGPCRs to adenylyl cyclase activation on endosomes.

Although the two above-mentioned studies strongly

support 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 of

multiple sclerosis[45]. Interestingly, Mullershausen et al.

have found that activated S1P1 receptors continue to

signal to Gi for hours, as shown by a persistent inhibition

of forskolin-stimulated cAMP production, in spite of con-

sistent internalization[12]. Moreover, analogs with lowerhydrophobicity but conserved potency and efficacy were

unable to promote persistent signaling. These findings

support the view that S1P1 receptors can continue to signal

to Gi and thus lead to persistent adenylyl cyclase inhibition

at intracellular sites. In contrast, the Gq-dependent acti-

vation of the PLCCa2+ signaling pathway appears

restricted to the cell surface.

Functional consequences of GPCRcAMP signaling on

endosomes

Although these new findings clearly support the existence

of GPCRcAMP signaling pathways on endosomes, our

understanding of their functional relevance is still limited.

What appears clear is that, differently from what is occur-

ring at the cell surface, GPCRcAMP signaling on endo-

somes is persistent. This phenomenon might be

particularly relevant in vivo, where the access to ligands

is often limited and can vary over time. This is the case for

several hormones, including TSH [46], that are secreted

with a circadian rhythm or for neurotransmitters, whose

pulsatile secretion results in submillisecond transients.

Thus, as anticipated by Miaczynska et al. [19], sustained

cAMP production from internalized receptors can provide a

memory mechanism, allowing cells to react with pro-

longed responses to short-term stimuli. As suggested by

the case of the PTHR, this type of intracellular signaling

might 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 might

have different outcomes than signaling from the plasma

membrane. In the case of the TSHR, signaling from inside

the cell appears to be more efficiently coupled to the PKA-

dependent reorganization of actin cytoskeleton, an event

that 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 cAMP

production makes sense only if cAMP cannot freely diffuse

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

intracellular compartments. A FRET reporter for cAMP (Epac1camps) [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, Gsis detected by immunofluorescence with a specific antibody.(c) Signalingfrom 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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inside cells. Indeed, although restricted cAMP diffusion

has been advocated to account for the specificity of certain

GPCR effects, whether or not cAMP appears to freely

diffuse seems to depend both on the cell type and on the

experimental 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 be

spatially restricted. Furthermore, the existence of cAMP

gradients is predicted on the basis of the spatial segre-

gation of adenylyl cyclases on the membrane and certain

phosphodiesterase isoforms in the cytosol [53]. In thisregard, GPCRcAMP signaling from intracellular sites

might provide a new basis to explain the activation of

targets located at distant sites, such as the nucleus, in

the presence of restricted cAMP diffusion.

Another example of how signaling from inside the cell

can differ from that occurring at the cell surface comes from

the above-mentioned study on S1P1 receptors. S1P1 recep-

tors are coupled to both Giand Gq. Thus, stimulation with

FTY720phosphate results in both inhibition of cAMP

production and increase of intracellular Ca2+ concen-

trations. However, although signaling from the plasma

membrane seems to be coupled to both pathways, signaling

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 selective

localization of effectors on different cellular membranes

(e.g. plasma membrane or endosomes), could provide the

basis for a temporal regulation of receptor coupling to

different downstream signaling pathways.

Concluding remarks

Based on these recent results we propose a new model of

GPCR signaling (Figure 4). Several GPCRs are interna-

lized together with their ligands (and perhaps with Gproteins and adenylyl cyclases) in endosomes or other

intracellular compartments. Here, at least some of them,

namely TSHR, PTHR and S1P1 receptors, find the machin-

ery required for cAMP production. As both ligands and

receptors remain confined in endosomes for some time, this

mechanism permits prolonged signaling even after

removal of the agonist from the extracellular space. In

addition, as suggested by the effects of internalized TSHR

on actin cytoskeleton, this new type of signaling can pro-

duce specific functional outcomes.

The advent of genetically encoded fluorescent reporters

for monitoring cAMP levels in living cells [42,5456]has

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

internalization together with their ligands into endosomes containing both Gsand 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 G iand 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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led to new insights into the mechanisms of GPCR signaling

and signal compartmentalization. Notably, the use of a

fluorescent cAMP reporter was instrumental for both find-

ings on TSHR and PTHR persistent signaling after intern-

alization. In addition, the availability of a transgenic

mouse with ubiquitous expression of a fluorescent cAMP

reporter allowed us to study TSHR signaling directly in

native thyroid follicles (i.e. very close to physiological

conditions). 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 that

might hinder our progress. First, the currently used

methods for inhibiting GPCR endocytosis (which were

applied with some success to study short-term responses)

appear inadequate to analyze long-term effects (such as

those on gene transcription), because they target molecules

such as clathrin or dynamin, which are implicated not only

in 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 limits

our capabilities of fully appreciating their subcellular

localization and interactions in physiological conditions.

Third, the endocytic compartment is highly intricate and

dynamic, 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 selective

strategies to inhibit GPCR endocytosis will most likely be

required to further advance our knowledge in this field.

Another issue to consider is that endosomes represent

only one of several possible sites of intracellular GPCR

signaling. For instance, some GPCRs, such as the chemo-

kine receptor CCR5 or the gonadotropin-releasing hormone

receptor, appear to be largely retained in the ER and Golgi

complex, even when observed in their natural context

[57,58]. In addition, there is evidence for early association

of GPCRs, G proteins and their effectors in these intracellu-

lar compartments[59]. As suggested by a recent study on

intracellularly retained vasopressin V2 receptor mutants,

such biosynthetic compartments might contain functional

GPCRs that can be activated by cell-permeable agonists

[60]. Thus, particular care should be taken when drawing

conclusions on the subcellular localization of GPCR-

initiated signals in response to lipophilic ligands.

With this in mind, there are several important questions

that can and need to be answered. First, is persistent GPCR

signaling to cAMP limited to a few receptors or is it a moregeneral phenomenon? And if it is a peculiar feature of

certain receptors only, what are the determinants? Indeed,

even if persistent cAMP signaling after internalization

might be common to other GPCRs, its contribution to the

overall signaling outcome might vary as a consequence of

complex interactions between factors suchas ligand affinity,

degree of receptor phosphorylation, affinity for b-arrestins

and 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 on

these considerations, it might not be by chance that this

phenomenon has been described for TSHR and PTHR, two

peptide/protein hormone receptors that form rather stable

complexes with theirligands. Second, can signaling through

other G proteins also occur at intracellular membranes?

Third, what is the physiological and pathophysiological

relevance of GPCR signaling at endosomes? For instance,

persistent TSHR signaling at endosomes might playa rolein

the pathogenesisof Gravesdiseaseor in disorders caused by

TSHR activating mutations. Fourth, what are its pharma-

cological implications? As already shown for S1P1 and PTHreceptors, different ligands can preferentially induce

plasma membrane or intracellular signaling, which can

be relevant for future drug design. Furthermore, interfering

with endocytosis might become a new tool for fine tuning

GPCR signaling and therefore a new strategy for thera-

peutic intervention.

Although further studies will be required to fully

appreciate 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 be

explored.

AcknowledgementResearch by the authors referred to in this publication is supported by

grants from the European Research Council (Advanced Grant TOPAS)

and the Deutsche Forschungsgemeinschaft (SFB487).

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