Signaling from endosomes: Location makes a difference

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1601 – 160 9

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Review

Signaling from endosomes: Location makes a difference Lukasz Sadowski, Iwona Pilecka, Marta Miaczynska⁎ International Institute of Molecular and Cell Biology, Laboratory of Cell Biology, 4 Ks. Trojdena Street, 02-109 Warsaw, Poland

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

In all transmembrane receptor systems the kinetics of receptor trafficking upon ligand

Received 15 September 2008

stimulation is maintained in a balance between degradative and recycling pathways in order

Accepted 23 September 2008

to keep homeostasis and to strictly control receptor-mediated signaling. Endocytosis is commonly

Available online 7 October 2008

considered as an efficient mechanism of uptake and transport of membrane-associated signaling molecules leading to attenuation of ligand-induced responses. Accumulating evidence, however,

Keywords:

shows that signaling from internalized receptors not only continues in endosomal compartments,

Endocytosis

but that there are also distinct signaling events that require endocytosis. Endocytic organelles

Signaling

form a dynamic network of subcellular compartments, which actively control the timing,

Receptors

amplitude, and specificity of signaling. In this review we provide examples in which signal

Internalization

transduction either requires an active endocytic machinery, or directly originates from various

Endosomes

types of endosomes. Based on recent discoveries, we emphasize the close interdependence

Trafficking

between signaling and endocytosis, and the physiological relevance of endocytic transport in health and disease. © 2008 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . Signaling is influenced by the route of internalization. Signaling from the early endocytic compartments. . . Signaling from the late endocytic compartments . . . Signaling from the recycling endocytic compartments . Functional implications of endocytosis in signaling . . Conclusions . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Signaling is a phenomenon by which a cell responds to stimuli from the surrounding environment. Receptors which are harbored in the plasma membrane are specific sensors mediating transfer of ⁎ Corresponding author. Fax: +48 22 597 07 26. E-mail address: [email protected] (M. Miaczynska). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.09.021

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information from the exterior to the cell interior by activating a cascade of downstream signaling molecules and triggering specific physiological outputs. However, signaling is not restricted just to the plasma membrane. It is known that many receptors that are internalized into endosomes in a process called receptor-mediated

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E XP E RI ME N TAL C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1601 – 160 9

endocytosis, are still potent for signal transduction as their activated tails remain exposed into the cytosol and can interact with other proteins [1–4]. In the case of receptor tyrosine kinases (RTKs), there are several events required for signal propagation upon ligand stimulation: the receptor changes its conformation, dimerizes and becomes phosphorylated, upon which a complex of signaling molecules assembles, thus initiating signaling cascades. Following activation by the ligand, receptors may enter the endocytic system via different pathways, e.g. clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE) [3,5]. Subsequently, they are directed to early endosomes and from there either sent to multivesicular bodies (MVBs)/late endosomes and lysosomes for degradation, or recycled back to the plasma membrane via recycling endosomes. During the endocytic transport, receptors may become dephosphorylated, ubiquitylated, and dissociated from the ligand, all of which contributes to signal attenuation. Changes in localization of the receptor can affect the composition of attached signaling molecules. Moreover, the sorting events that lead receptors to degradation or recycling determine the overall level of available receptors, influencing the cellular response to the stimuli. According to the latest reports it is clearly noticeable that the diverse internalization pathways of receptors and their intracellular trafficking via distinct endocytic structures control the strength and duration of signals and provide an environment for interactions of receptors with different signaling molecules [3–5]. Here we will review the roles of individual endocytic compartments in signal transduction.

Signaling is influenced by the route of internalization It is generally accepted that internalization of receptors following ligand stimulation is important for proper signal transduction. The primary role for such clearance of receptors from the cell surface is to regulate the number of available receptors. However, in many cases the internalization has another, equally important role: to transport the ligand–receptor complexes into the cell interior for further signal propagation. Early indications for the role of compartmentalization in ligand-mediated receptor signaling came from biochemical analysis identifying signaling molecules associated with activated epidermal growth factor receptor (EGFR) in endosomal fractions [6]. Further evidence was provided upon the usage of a dominant-interfering mutant of dynamin (K44A) [7]. Dynamin is crucial for fission of clathrin-coated pits from the plasma membrane and creation of clathrin-coated vesicles (CCV). Blockage of internalization by expression of K44A/ dynamin resulted in enhanced cell proliferation and hyperphosphorylation of phospholipase-Cγ and adaptor protein SHC in cells stimulated with epidermal growth factor (EGF). On the other hand, endocytic trafficking was required for the full phosphorylation of EGFR, the 85 kD regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinases (MAPKs) ERK1/2 [7]. Similarly, K44A/dynamin selectively modified signal transduction downstream of insulin receptor, inhibiting the activation of SHC, ERK1/2 and the PI3K pathways without affecting insulin-stimulated receptor autophosphorylation, activation of insulin receptor substrate 1 (IRS1) or AKT [8]. Furthermore, interfering with endocytosis by means of

K44A/dynamin or mutated Rab5 demonstrated that endocytic internalization and recycling were specifically required for H-Ras but not K-Ras signaling through the Raf/MEK/MAPK cascade [9]. All these cases indicated that different sets of signaling molecules are activated on the plasma membrane and on endosomes. Indeed, nerve growth factor (NGF)-stimulated TrkA receptors present at the plasma membrane were reported to transiently activate Ras, whereas receptor complexes present on endosomes selectively mediated sustained activation of Rap1 and MAPK [10]. Also for EGF, biochemical isolation of protein complexes associated with ligand-stimulated EGFR demonstrated that the receptor remained active in the early endosomes but the composition of endosomal and plasma membrane-bound signaling complexes partially differed [11]. Interestingly, it appears to be a common phenomenon for a number of transmembrane receptors that their mode of internalization may impact the signaling process and the final cellular response. Parallel to CME, there are clathrin-independent entry pathways that are also involved in cell signaling (Fig. 1). Certain molecules can be internalized by caveolae, which are plasma membrane invaginations enriched in glycosphingolipids, cholesterol and protein caveolin-1 [12]. Among many postulated functions, caveolae were also proposed to sequester receptors on the plasma membrane and thereby preventing their overactivation, as reported for two RTKs: EGFR and platelet-derived growth factor (PDGF) receptor. Cells pre-treated with EGF did not respond to the subsequent application of PDGF, because the PDGFR was sequestered in caveolae. The same mechanism was observed in case of EGFR in cells previously treated with PDGF [13]. There are convincing examples showing that the route of internalization controls the biological outcome as well as the extent and duration of downstream signaling. It was first reported for transforming growth factor β (TGFβ) receptors, which can be internalized both via CME and caveolae. While the former pathway leads to activation of downstream effectors on early endosomes (see below), the latter leads to rapid degradation of TGFβ receptors and termination of their signaling via inhibitory Smad7 and Smurf2 [14]. Recent studies on other ligands of TGFβ family such as activin/Nodal identified Rap2 GTPase as a key regulator of receptor trafficking, which directly affects signaling in Xenopus embryogenesis. In the absence of ligand, Rap2 directs activin/Nodal receptors towards recycling to maintain their levels on the plasma membrane, whereas upon ligand stimulation receptor degradation is delayed thus promoting prolonged signaling [15]. The preferred pathway of internalization and further endocytic trafficking may be influenced by the abundance of a ligand, as demonstrated for EGFR [16,17]. When stimulated by low amounts of EGF, the receptors are internalized via CME and this pathway leads to the increased recycling of the receptor, thus contributing to prolonged signaling. Upon cell stimulation with high doses of EGF, in addition to CME, the receptor is internalized via ubiquitindependent CIE, which does not increase signal propagation but only enhances the receptor degradation rate. Interestingly, CME appears to be indispensable for maintaining the proper duration of EGFR signaling leading to the final biological responses such as DNA synthesis. Similar dependence between the concentration of a ligand, its internalization route and the signaling output has been also recently reported for PDGF [18]. Cells stimulated with low doses of PDGF respond with cytoskeleton rearrangements and migration, which depends on the activity of CME. When PDGF


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Fig. 1 – Schematic representation of various endocytic compartments with the examples of signaling molecules acting locally on the surface of intracellular organelles. See text for detailed explanations.

concentration is increased, the signaling output is shifted to the induction of mitogenesis and cell proliferation, which relies on raft/caveolin pathways. These studies have likely uncovered a more general principle by which cells could exploit different internalization routes to sense the amounts of a ligand and to control cellular responses subsequent to stimulation with different ligand concentrations. Such a concept based on EGF endocytosis has been recently described in a mathematical model [19]. Other RTK members, such as neurotrophin receptors TrkA and TrkB can also be internalized via different routes. Both TrkA and TrkB can undergo CME when activated by their respective ligands: NGF and brain-derived neurotrophic factor (BDNF). Interestingly, while TrkA is predominantly sorted to recycling, TrkB is mainly degraded in lysosomes – and these different endocytic fates of Trk receptors are correlated with various signaling events and biological responses [20]. On the other hand, both Trk receptors

can also be internalized into macroendosomes (see below) in a process of clathrin-independent macropinocytosis at plasma membrane ruffles, which requires Rac GTPase, dynamin and the trafficking protein Pincher [21,22]. Moreover, endocytic internalization and trafficking are important for signaling downstream of receptors other than RTKs. Recent studies demonstrated that LRP6 co-receptor for Wnt ligands can be endocytosed via two distinct mechanisms. Wnt3a stimulates caveolin-dependent internalization of LRP6 leading to stabilization of β-catenin and activation of signaling, whereas Dickkopf antagonizes this process by diverting LRP6 away from the caveolincontaining fractions and inducing its endocytosis via clathrindependent pathway thus preventing β-catenin signaling [23]. Spatial compartmentalization of active receptors between the plasma membrane and the endosomes can result in opposite physiological outputs. Immediately upon ligand stimulation,

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tumor necrosis factor receptor 1 (TNFR1) shows capability of signaling from the plasma membrane, where it promotes prosurvival signals via nuclear factor-κB (NF-κB). After TNFR1 internalization by CME, which occurs in the range of minutes after ligand stimulation, NF-κB signaling is terminated and the pathways activating caspase-8 leading to apoptotic cell death are initiated [24]. Similar spatial and temporal compartmentalization of non- and pro-apoptotic signaling initiated from the plasma membrane and endosomes, respectively, was proposed for CD95/ Fas receptor [24]. In these cases, endocytic internalization would provide a key switch mechanism between two different signaling pathways employing distinct biochemical components and leading to opposite biological responses.

Signaling from the early endocytic compartments Early endosomes serve as the first sorting station following CME: from there cargo is either directed into MVBs/late endosomes and lysosomes for degradation, or it is recycled back to the plasma membrane. Canonical early endosomes are marked with the small GTPase Rab5 and early endosome antigen-1 (EEA1). The receptors internalized into early endosomes reach here the biochemical environment distinct from that of the plasma membrane, but nevertheless enabling interactions between signaling molecules, some of which are endosome-specific (Fig. 1) [1,4]. The concept of signaling endosomes originates largely from studies in neurons and has been reviewed elsewhere [25,26]. Due to the long distances between the axon terminals and the cell bodies it was difficult to explain the range and speed of retrograde signal transduction by simple diffusion of signaling molecules. By tracking internalized NGF it was shown that both CCVs and subsequently early endosomes are carriers of activated TrkA receptors interacting with signaling molecules of ERK1/2 and PI3K/AKT pathways [27,28]. Moreover, such microtubule- and dynein-based transport of endosomes is required for neuronal survival [29–31]. As reported recently, an alternative route of NGFTrkA internalization by macropinocytosis leads to formation of large structures termed macroendosomes (see above). Interestingly, although macroendosomes are positive for early endosomal marker Rab5 and lack late endosomal Rab7, they have multivesicular morphology typical for late endosomes [21,22]. They appear long-lived and resistant to degradation, and were proposed to sustain NGF signaling over prolonged times. Further evidence for signaling capacity of receptors localized to early endosomes was provided by a system where reversibly inhibited receptors were internalized and subsequently activated on endosomes. Such active endosomal EGFR and PDGFR recruited signaling molecules and evoked physiological responses like cell proliferation and migration [32,33]. Signaling from early endosomes is also exploited by receptors of the TGFβ family, such as receptors for activins, bone morphogenetic proteins (BMPs), Nodal and TGFβ itself. Upon activation by extracellular ligands these receptors phosphorylate Smad transcription factors, inducing their translocation to the nucleus. It turned out that Smad2/3 activation requires a specific interaction with the adaptor protein SARA (Smad anchor for receptor activation) that predominantly localizes to early endosomes via its FYVE domain [34]. This zinc finger domain binds to phosphatidylinositol 3-phosphate that is enriched on the membrane of early endosomes [35]. Mislocalization of SARA

by mutations in its FYVE domain caused impaired activinA signaling in endothelial cells, pointing to the importance of early endosomes for proper signal propagation [36]. Interestingly, the interaction of the TGFβ receptor with another early endosomal adaptor containing FYVE domain, endofin, promotes phosphorylation and nuclear translocation of Smad4 [37]. In addition, endofin is required for Smad1-mediated signal propagation downstream of BMP receptor [38]. Functional importance of signaling from early endosomes has been recently reinforced by studies on Rac-mediated cell migration [39]. Upon stimulation with hepatocyte growth factor (HGF), its receptor is internalized via CME into early endosomes. Such internalization into Rab5-positive structures is necessary to activate Rac GTPase via its endosome-associated guanine nucleotide exchange factor (GEF) Tiam1. Subsequent Arf6-mediated recycling of active Rac to the plasma membrane is required for localized formation of migratory protrusions [39]. Post-translational modifications of signaling molecules specific for early endosomes or occurring at the level of early endosome represent yet another mechanism of endosomal signaling. It is well known that in the process of receptor downregulation, sustained ubiquitylation of RTKs marks them for degradation. A recent report demonstrated an endosome-specific ubiquitylation event occurring in the TNF-α pathway via ubiquitin ligase CARP-2 containing the FYVE finger domain [40]. Upon TNF-α stimulation, TNFR1 assembles in a complex with signaling molecules TRAAD, RIP and TRAF-2. RIP is required for NF-κB activation, but its prolonged signaling may lead to chronic inflammatory responses. Endosomal CARP-2 polyubiquitylates RIP and targets it towards proteasomal degradation, thereby negatively regulating TNF-α signaling. The heterogeneity of the endosomal system can be exploited in order to provide spatial regulation of intracellular signal propagation. A separate subpopulation of early endosomes bearing Rab5 effectors APPL1 and APPL2 was reported to be distinct from the canonical EEA1-positive early endosomes and required for mitogenic signaling [41]. Importantly, subsequent studies in zebrafish demonstrated that APPL1 localized to endosomes regulates AKT activity and its substrate specificity, controlling GSK-3β but not TSC2, which is necessary to support cell survival during development [42]. These studies indicate that several populations of endosomal compartments can act as platforms for assembly of different sets of signaling effectors. A number of specialized endosomal compartments have been described for example in polarized cells [43] and they could potentially be involved in compartmentalization of signaling. In developing fly wing epithelium, a subpopulation of apical endosomes bearing TGFβ-like ligand Dpp, its receptor and SARA associate with the mitotic spindle to ensure proper distribution of signaling molecules between daughter cells [44]. Finally, yet another direct link between endosomes and nuclear signaling events appears to be provided by endosomal proteins which are able to translocate themselves to the cell nucleus, of which APPL proteins represent one example. Such moonlighting endocytic adaptors and their nuclear functions have been recently reviewed [45].

Signaling from the late endocytic compartments Following passage through early endosomes, receptors directed towards degradation reach another sorting station: MVBs or late


endosomes, which constitute morphologically distinct large structures with intraluminal vesicles and are marked with Rab7, lysosomal-associated membrane protein-1 (LAMP1), and lysobisphosphatidic acid [46]. Sorting of receptors marked with ubiquitin at their cytoplasmic domain occurs at the limiting membrane of MVBs thanks to the highly organized sequential action of several multiprotein complexes called ESCRTs (endosomal sorting complex required for transport). Initially, HGF-regulated tyrosine kinase substrate (Hrs) binds to ubiquitylated receptors, followed by recruitment of ESCRT components that are required for the incorporation of receptors into intraluminal vesicles leading eventually to their lysosomal degradation [47]. Indeed, depletion of Hrs causes increased recycling and strong inhibition of degradation of EGFR concomitant with prolonged ERK signaling [48,49]. Receptors entrapped in intraluminal vesicles have no signaling capabilities as their intracellular domains no longer face the cytosol, thus such mechanism leads to signal downregulation. On the other hand, back-fusion of intraluminal vesicles with the limiting membrane of MVBs was demonstrated to occur during MHC class II processing in immune cells [50] or for the release of viral nucleocapsids [51]. Although there is currently no evidence for the return of signaling receptors sequestered in the intraluminal vesicles back to the limiting membrane of MVBs facing cytosol, such possibility cannot be formally excluded, which would endow late endosomes with yet another regulatory mechanism of signal propagation. Although late endosomes generally seem to be the place of signal attenuation, these organelles can still contain activated receptors and their downstream effectors [52] and play a role in active signal transduction via compartment-specific interactions. In case of the MAPK pathway, late endosomal adaptor p14 complexed to MP1 serves as a scaffold recruiting MEK1 to the membranes, and these interactions are important for proper ERK signaling [53,54]. Strikingly, the p14-MP1 complex mistargeted to the plasma membrane is not active in terms of potentiating MAPK activity [53]. In addition to regulating MAPK signaling, p14 appears to function in the biogenesis of late endosomes, as shown by recent findings describing human primary immunodeficiency syndrome caused by a mutation in p14 [55]. Another example of active signal propagation from late endosomes involves a guanine nucleotide exchange factor for Rap1, PDZ-GEF1, which interacts with TrkA receptor at late endosomes and mediates sustained Rap1 and ERK signaling necessary for neurite outgrowth [56]. Furthermore, a late endosomal protein spinster is involved in the TGFβ signaling leading to synapse development [57] and other studies have implicated Rab7-positive endosomes as the long-distance signaling carriers in retrograde transport of neurotrophins [58]. Interestingly, there is also an example of G protein-coupled receptor (GPCR) termed OA1 (for ocular albinism 1) which resides not on the plasma membrane, but on late endosomes/lysosomes and melanosomes [59], arguing for a signal transduction system operating intracelullarly at the level of endosome- and lysosome-related organelles. Properties of late endosomes appear to be dependent not only on protein composition or pH but also on their localization within the cell. Mistargeting of late endosomes to the cell periphery caused slower degradation and prolonged signaling of EGFR, resulting in the sustained phosphorylation of ERK and p38 MAPK which led to the hyperactivation of Elk1 transcription factor. On the other hand, late endosomes forced to cluster tightly around the nucleus exhibited a delay in receiving EGFR which resulted in a

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sustained activation of ERK but not of p38, with the final negative impact on Elk1 activity [60]. Also in case of early endosomes, their mislocalization to the cell periphery by modulating the activity of kinesin KIF16B slowed down EGFR degradation, while clustering them at the perinuclear area downregulated recycling but accelerated degradation of EGFR [61]. These examples demonstrate that intracellular localization of distinct endocytic compartments is essential for both proper trafficking and signaling.

Signaling from the recycling endocytic compartments Receptors can be recycled in two ways: fast via peripheral early endosomes marked with Rab4, and slow via perinuclear recycling compartment marked with Rab11 [62]. Endocytic recycling is another way by which the cell controls levels of plasma membrane receptors. In the steady-state, the pool of plasma membrane receptors is internalized and recycled to the plasma membrane at a constant rate, independent of ligand stimulation. In case of GPCRs their activity is highly controlled by recycling although they can also undergo lysosomal degradation [63]. Desensitization of GPCRs is mediated by their phosphorylation and internalization, the latter mediated by adaptor proteins β-arrestins 1 and 2. By binding to the phosphorylated receptor arrestins mediate dissociation of the G protein, and by binding to clathrin and the adaptor protein AP-2 they initiate internalization of the receptor. Internalized receptors reach early endosomal compartment, where they become dephosphorylated and dissociate from their ligands. Final resensitization of GPCRs is achieved by directing the receptors to the plasma membrane via recycling endosomes. Interestingly, some GPCRs like β2-adrenergic and μ opioid receptors undergo Hrs-mediated recycling, indicating that the function of Hrs on endosomes is not limited to MVB formation [64]. Generally, in case of GPCRs endocytosis is used either for signal attenuation or for restoring sensing capability of a cell. Importantly, β-arrestins serve as scaffolds mediating the interaction between GPCRs and components of MAPK cascades [65,66], arguing for an active signaling events occurring during the receptor transit in endosomal compartments [4]. Interestingly, β-arrestins represent another example of endocytic adaptors capable of translocating to the nucleus where they can associate with transcription factors, affecting gene expression and histone acetylation in response to GPCR stimulation [67,68]. The importance of recycling endosomes for transmitting signals has been demonstrated in studies on determination of cell fate in the developing sensory organ in Drosophila [69]. This process requires asymmetric divisions of precursor cells and is regulated by Notch/Delta signaling. Intriguingly, the asymmetry between daughter cells is generated by differential trafficking of ligand Delta via Rab11-positive recycling endosomes: in pIIa cell Delta is recycled in a Rab11-dependent manner which is essential for its activity, while in pIIb cell recycling endosomes do not form (due to the inactivation of Rab11 effector Nuclear fallout), Delta is not recycled but rather becomes degraded. In mammalian cells, a shift in PDGFR-β trafficking from degradation towards recycling was shown to depend on T-cell protein tyrosine phosphatase TC-PTP which normally dephosphorylates the receptor after internalization. Loss of TC-PTP induced increased recycling of PDGFR-β via the Rab4 recycling compartment [70], providing the first evidence for a tyrosine

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phosphatase modulating growth factor receptor trafficking. It will be interesting to see whether defects in hematopoiesis and immune function of TC-PTP knockout mouse [71] can be linked to the enhanced recycling of signaling receptors. Recycling endosomes appear also to be involved in compartmentalization of Ras signaling. It was shown that H-Ras, but not K-Ras, dynamically interacts with Rab11-positive endosomes (thanks to its palmitoylated C-terminus) which was postulated to contribute to its signaling specificity [72].

Functional implications of endocytosis in signaling Regulation of signaling by endocytosis has functional importance in many biological and physiological processes. Signaling following the activation of growth factor receptors often leads to cell proliferation, and any disturbance of the sorting system may promote overactivation of signaling leading to uncontrolled cellular divisions. One of the control mechanisms is ubiquitylation of the intracellular tail of the receptor upon ligand activation, which directs the activated receptor towards degradation. Both overactivation and impaired downregulation of RTKs are associated with carcinogenesis [73]. In the case of the EGFR family members (e.g. ErbB2 that is often overexpressed in breast and ovarian malignancies), their escape from degradative pathway towards recycling leads to transformational changes [74]. Dysregulation of trafficking of other RTKs, like PDGFR or Met, may also bear oncogenic potential [75]. In addition, endocytosis plays a role in cell migration and invasion mediated by integrins which regulate contacts between cells and the extracellular matrix. Functions of integrins and their signaling leading to cell migration are strictly controlled by their endocytic trafficking [76]. Disturbance of endocytic trafficking is also implicated in severe neurodegenerative disorders like Alzheimer disease (AD) which is characterized by deposition of amyloid β (Aβ) plaques in brain. Endosomes appear to be one of the major sites of Aβ generation and several reports indicate that altered endocytic trafficking may play role in development of AD [77]. For instance, the neuronal sortilinrelated receptor SORL1 directs trafficking of amyloid precursor protein to recycling and diverts it from accumulation in the compartments generating Aβ [78]. Another example of impaired trafficking resulting in pathogenic conditions is Huntington's disease. It is characterized by mutated protein huntingtin, which localizes to endocytic vesicles and affects membrane trafficking in neurons leading to neurodegenerative changes [79]. Endocytosis participates in the induction of immunological responses by antigen presenting cells. Dendritic cells and macrophages internalize antigens, process them proteolytically within the endocytic pathway, and subsequently recycle the antigen fragments back to the plasma membrane to present them bound to MHC class II complexes [80]. Another kind of cellular self-defense is achieved by innate immunity which is maintained by Toll-like receptors (TLR). They have capability to initiate immune responses upon activation by extracellular stimuli like nucleic acids, lipids or proteins of viruses and bacteria. TLR3, 7, 8, 9 are localized predominantly on endosomes and require internalization of ligands to initiate signaling [81], thus providing another example of an active role of endosomes in signal transduction. In contrast, TLR1, 2, 4, 5, 6 reside on plasma membrane, but for example upon binding of lipopolysaccharide (LPS) TLR4 is internalized into early

endosomes and undergoes degradation, thus preventing sustained anti-inflammatory response mediated by LPS [82]. On the other hand, many pathogens like viruses, bacteria and parasites hijack endocytic pathways for their own benefit [83]. There are an increasing number of reports documenting the engagement of endocytosis in certain developmental processes, reviewed in details elsewhere [84,85]. For example, spreading of some morphogen gradients (e.g. Dpp in developing Drosophila wing) requires active endocytic transport [86]. In the case of activin gradient in Xenopus, the duration of endocytic transport of activated receptors before their degradation determines the intracellular concentration of active Smad2, which is important for correct gradient interpretation and the choice of cell fate [87]. Moreover, internalization, endosomal trafficking and proteolytic modifications are crucial for signaling mediated by Notch receptors and their membrane-bound ligands of Delta/Serrate/Lag2 family which regulate multiple processes during development and adult life [88]. Asymmetry in Notch signaling between two interacting cells is due in part to differences in endocytic sorting and transport routes operating in ligand-expressing and receptor-expressing cells [89]. Eph receptors, a subfamily of RTKs involved in development of the nervous system, also recognize membranebound ligands (ephrins) present on neighboring cells, thus establishing cell–cell contacts. These interactions trigger a bidirectional endocytosis where the ephrin-containing cell is internalizing Eph receptor, and vice versa. This complicated mechanism leads to the activation of Rho GTPases and changes in actin cytoskeleton that result in repulsion or attraction of contacting cells. Involvement of endocytosis in Eph receptor/ephrin signaling is currently investigated, but there are indications that it plays a critical role in the functionality of these receptors [90]. Beside the animal world, plants have also adapted endocytosis for signal transduction evoked by phytohormones like auxins and brassinosteroids which are responsible for plant development and growth [91]. Auxin regulates internalization and recycling of its transporter PIN1 [92], while brassinosteroid signaling is enhanced upon increasing the endosomal pool of BRI1 receptor [93].

Conclusions All data documenting links between endocytosis and signaling strongly indicate that these two processes are inseparable and mutually interdependent. Their interplay was proved for various classes of receptors, in different cell types, and in distinct physiological processes. Endocytosis seems to control signaling events both spatially and temporally, as it provides a hierarchical network that compartmentalizes signaling molecules for either signal propagation or attenuation. Disruption of that highly ordered system may contribute to carcinogenesis and other diseases. Growing evidence suggests that, in addition to wellestablished signal initiation from the plasma membrane, signaling from endosomes can provide both quantitative and qualitative inputs to signal propagation. Endosomes seem to fulfill at least three distinct signaling functions, depending on the receptor system and cellular context. First, they ensure temporal prolongation of signal transduction initiated upon ligand binding at the plasma membrane and continued after internalization. Second, they act as scaffolds to generate specific signals through assembly of unique signaling complexes due to particular composition of


endosomal membranes. Third, thanks to their cytoskeleton-based motility they deliver active signaling molecules to various locations within the cell. In many cases, the precise mechanisms responsible for the variety of physiological outputs of endocytic processes still remain to be discovered. It seems that the classical distinction between early, late, and recycling endosomes may not be sufficient to support the factual variability of specialized endosomal subpopulations that contribute to differential signaling outputs. In addition, some studies raise a question whether the recycling of growth factor receptors is not a largely underestimated player regulating the extent of signal activation. Even less is known about intermediates and regulatory processes in the endocytic pathways independent of clathrin. Future discoveries are likely to put further emphasis on the importance of compartmentalization and local specialized domains for integrating the multitude of signaling processes within the endocytic system.

Acknowledgments We thank Drs. Kaisa Haglund and Beata Pyrzynska for critical reading of the manuscript. Research in Miaczynska's laboratory is supported by an International Research Scholar grant from the Howard Hughes Medical Institute, a Senior Research Fellowship from the Wellcome Trust (076469/Z/05/Z), by the European Union LSHG-CT-2006-019050 (EndoTrack), Polish Ministry of Science and Higher Education (2P04A03828 and Mobilitas.pl network) and Max Planck Society (Partner Group programme).

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