Intracellular trafficking of hormone receptors

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1 Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August 2004 Intracellular trafficking of hormone receptors Zsuzsanna Gáborik and László Hunyady Department of Physiology, Semmelweis University, Faculty of Medicine, H-1088 Budapest, Hungary Agonist binding stimulates endocytosis of hormone receptors via vesicular uptake mechanisms. Interactions of the intracellular domains of receptors with specific targeting proteins are crucial for sorting of internalized receptor in endosomes. Some receptors are targeted for very rapid (e.g. b2-adrenergic receptor) or slower (e.g. AT 1 angiotensin receptor) recycling pathways, whereas others are targeted to lysosomes for degradation (e.g. EGF receptor or PAR1 protease-activated receptor). This review discusses the mechanisms involved in these processes, which regulate surface receptor expression and set the stage for intracellular signaling of G protein-coupled and growth factor receptors. The number of cell surface receptors is an important determinant of the hormonal responsiveness of tissues. Steady state levels of plasma membrane receptors are determined by the balance between pathways that deliver receptors to the cell surface and those that remove them by endocytosis. Receptor endocytosis occurs via diverse vesicular uptake mechanisms. Recent experimental data have begun to elucidate the mechanisms that determine the fate of internalized receptors. After intracellular processing in endosomal compartments, the internalized receptors either recycle back to the plasma membrane to restore the plasma membrane receptor pool, or enter compartments where the receptor molecule is degraded (e.g. lysosomes). Initially it was generally assumed that receptor internalization contributes to desensitization by reducing the number of available surface receptors. Although this model is still valid for tyrosine kinase receptors, many G protein-coupled receptors (GPCRs) are desensitized at the cell surface by receptor kinases, and internalization allows dephosphorylation and resensitization of the receptor in intracellular compartments [1]. Recent data also suggest that signaling of many internalized receptors continues in endosomes. Signaling of internalized growth factor receptors was shown to continue after internalization, and association of GPCRs and activated mitogen-activated protein kinase signaling complexes in endosomes has also been reported [2]. Intracellular trafficking pathways Receptor endocytosis In most cases, endocytosis of hormone receptors is accelerated by agonist binding, whereas nutrient receptors, such as Corresponding author: László Hunyady (hunyady@puskin.sote.hu). Available online 4 July 2004 LDL receptor (LDL-R) and transferrin receptor (TfR), are endocytosed constitutively at a rate independent of ligand occupancy [3 5]. The best-identified pathway of receptor endocytosis is mediated by clathrin-coated vesicles (CCVs), first identified as the mechanism of LDL-R endocytosis [4,5]. TfR, EGF receptor (EGFR) and many GPCRs also internalize via CCVs [3 5]. In yeast monoubiquitylation regulates the internalization of pheromone receptors [6] but available data indicate that internalization of mammalian GPCRs is independent of receptor ubiquitylation [7 9]. Clathrin-mediated internalization of mammalian GPCRs frequently requires agonist-induced interaction of b-arrestin proteins with the receptor because in addition to their role in receptor desensitization, b-arrestins also serve as adapters linking the receptor to the endocytotic machinery [10,11]. This clathrin-mediated endocytosis requires the function of dynamin GTPases for vesicle formation and scission from the plasma membrane [12]. Internalization of receptors also occurs via non-coated vesicles, such as flask-shaped caveolae, and other pinocytotic mechanisms (Figure 1) [5,12]. Internalization via caveolae also requires dynamin; however, dynaminindependent endocytotic mechanisms have also been suggested [12,13]. Operation of different endocytic pathways of GPCRs in the same cell was first demonstrated in studies on the mechanism of internalization of cholecystokinin receptors [14]. Studies on dopamine receptors also support this hypothesis because D1 and D2 receptors (D1R and D2R) internalize in separate vesicles in the same cell [15]. For endothelin A receptor (ETA-R) the default pathway is endocytosis via caveolae, but cholesterol oxidation switches the internalization pathway from caveolae to CCVs [16]. A recent study suggested that internalization pathways of the b1-adrenergic receptor are primarily determined by the kinase that phosphorylates the receptor: protein kinase A-mediated phosphorylation directs the receptor to caveolae, whereas GPCR kinase (GRK)-mediated phosphorylation directs it to CCVs [17]. Internalization of the AT 1 angiotensin receptor (AT 1 R) occurs predominantly with a b-arrestindependent, clathrin-mediated pathway at physiological hormone concentration, but at high levels of receptor occupancy a b-arrestin-independent mechanism was also demonstrated [18,19]. The mechanism of endocytosis could have a major effect on the fate of internalized receptors. For example, TGF-b receptors internalized via caveolae are targeted for degradation, whereas those internalized via CCVs promote signaling [20] /$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi: /j.tem

2 Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August Clathrin-mediated endocytosis Early sorting endosome β-arrestin? Caveosome TRENDS in Endocrinology & Metabolism Compartments involved in intracellular trafficking of membrane receptors Internalized membrane receptors are first targeted to early sorting endosomes. Endosomes have a central role in intracellular trafficking and comprise a system of heterogeneous compartments that have been originally characterized as early and late, depending on the kinetics of the appearance of internalized proteins in these compartments [21]. Different vesicular organelles are differentiated by their morphological appearance, association with the appropriate Rab GTPases, different protein and/or lipid composition and, in some cases, by different luminal ph (Box 1). Early sorting endosomes In higher eukaryotic cells, CCVs lose their coats shortly after endocytosis and fuse with each other and/or with sorting endosomes. Sorting endosomes are located close to the cell periphery and have tubular structure, which resembles that of recycling endosomes (see below) [5]. However, these early endosomes can also contain multivesicular late endosome-like membrane invaginations. These tubular and multivesicular regions can therefore be considered the trans face of the organelle, and the central cisternal region functions as the entry or cis region [22]. The sorting endosome is a dynamic compartment and? Caveolinmediated endocytosis Clathrin- and caveolinindependent endocytosis Dynamin Caveolin Clathrin AP-2 Figure 1. Pathways of receptor-mediated endocytosis. The most well-characterized pathway of endocytosis is clathrin-coated pit formation and dynamin GTPasedependent scission of coated vesicles (w120 nm) from the plasma membrane. Clathrin-mediated endocytosis of G protein-coupled receptors (GPCRs) can occur with b-arrestin-dependent and independent mechanisms. Caveolae are flaskshaped invaginations of cholesterol and sphingolipid-rich plasma membrane with a diameter of w55 60 nm. This structure is coated by the dimeric protein caveolin that binds cholesterol. Caveolae can pinch off to form vesicles and this process also requires the function of dynamin. Clathrin- and caveolin-independent endocytosis also play a role in receptor-mediated endocytosis. The formation of these non-coated vesicles (w90 nm) can occur with dynamin-dependent or -independent mechanisms. These pathways are indicated by question marks in Figure 1. In addition to mechanisms of hormone receptor endocytosis shown in the figure, other vesicular uptake mechanisms (e.g. phagocytosis, macropinocytosis) also exist [5,12]. accepts input from both clathrin-mediated and fluid-phase endocytosis [13]. Membrane proteins and cargo are sorted from this compartment to different locations; hence these endosomes represent the single entry point for internalized receptors [22]. Recycling receptors are rapidly segregated away from their ligand after receptor-ligand uncoupling as a consequence of mildly acidic luminal ph (ca 6.0) in sorting endosomes. By contrast, persistent ligand occupancy in endosomes favors receptor sorting to lysosomes. Receptors can recycle back to the plasma membrane directly from early endosomes, or exit from this compartment in the direction of late endosomes or recycling endosomes [5,21,23]. Trafficking of many GPCRs to early endosomes has been shown by colocalization with markers of this compartment, such as transferrin, early endosome antigen 1 (EEA1) or Rab5 GTPase [24 27]. ETA-R and ETB-R are both colocalized with Rab5-positive endosomes, even though ETA-R recycles to the cell surface and ETB-R is targeted to lysosomes [27]. The Rab5 GTPase is required for processing of early endosomes, but studies with GTPase deficient mutant Rab5 demonstrated that it is also required for the endocytosis of several GPCRs [e.g. m4 muscarinic acetylcholine receptor (m4achr), b 2 adrenergic receptor (b 2 AR), D2R] [24,26,28]. Although Rab5 directly interacts with the C-terminal tail of the AT 1 R, internalization of this receptor is independent of the GTPase activity of Rab5, but it is still targeted to Rab5-containing early endosomes [29,30]. Interaction with Rab5 retained the receptor-b-arrestin complex in Rab5-positive early endosomes, therefore inhibiting the further processing of the AT 1 R [31]. Recycling pathways The main routes of receptor recycling and degradation are well separated, both topologically and functionally (see Box 1). TfRs recycle efficiently to the plasma membrane via fast and slow routes, which correspond to at least two separate transport steps, each with different molecular machinery. The rapid phase of TfR recycling requires Rab4, and is directed from Rab4- and Rab5-positive early sorting endosomes to the plasma membrane. The slow phase of TfR recycling is initiated from tubular extensions of sorting endosomes and reaches indirectly the plasma membrane through Rab11-positive perinuclear recycling endosomes. Recycling endosomes are tubular structures found close to the centrioles in some cell types and are less acidic than early endosomes [21,22,32]. EGFR is mainly excluded from recycling pathways, as it is predominantly targeted for degradation. GPCRs have individual trafficking patterns characteristic for each receptor (Figure 2). Although some GPCRs [e.g. protease-activated receptor 1 (PAR1)] are predominantly targeted toward lysosomes for degradation, many of them recycle back to the plasma membrane to replenish the biologically active cell surface receptor population. GPCRs can use different recycling pathways, as it was demonstrated by colocalization with different recycling compartments or inhibitory effects of mutant Rab proteins. Rapid recycling is considered to be the default pathway and is facilitated by dissociation of b-arrestin (e.g. b2ar, see below Class A receptors). These receptors

3 288 Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August 2004 Box 1. Molecular architecture of intracellular organelles involved in membrane trafficking Common steps in intracellular trafficking pathways include membrane budding to form vesicles, transport to a particular destination, and ultimately docking and fusion with the target membrane. Specificity of vesicle targeting is rendered in part by associated small GTPases of the Rab family. Substantial evidence has accumulated that most Rab proteins regulate the targeting/docking/fusion processes and that some of them regulate the budding process. Rab mutants are often characterized by a massive accumulation of certain vesicles in the respective pathway. Therefore GTPases of the Rab family are considered organelle markers owing to their restricted distribution. Although individual regulatory components in the endocytic pathway might be loosely distributed throughout several compartments, a particular combination is unique to each compartment. Active Rab5 is important for endocytosis via clathrin-coated pits and subsequent fusion of vesicles with early endosomes. The presence of Rab5 on early endosomes is also essential for their homotypic fusion. However, Rab5 is also associated with the plasma membrane. In addition to Rab5, early endosomes contain Rab5 effectors and regulator proteins, including early endosome antigen 1 (EEA1), rabaptin 5, rabenosyn-5 PtdIns3P and partners [70]. Molecules can exit early endosomes via several pathways. A direct pathway for recycling receptors to the plasma membrane depends on Rab4, referred to as fast recycling. Recent findings also implicate Rab4 in recycling via recycling endosomes, referred to as slow recycling route. It should be noted that it is likely that Rab5 and Rab4 act together to control influx into and efflux out of early endosomes respectively, because the two proteins exhibit concerted effector binding. A role for Rab11 has been documented in late recycling of transferrin receptor through the recycling endosomes. Indeed Rab11 is concentrated on recycling endosomes, and is proposed to regulate the slow return of recycling receptors to the plasma membrane. Proteins destined for degradation are delivered to late endosomes and then to lysosomes. This process is strongly inhibited by dominant negative Rab7, indicating that Rab7 is essential for transport from early to late endosomes. However it remains to be elucidated whether Rab7 is also required for transport from late endosome to lysosome [32]. Rab proteins also participate in the vesicular transport mechanism between other organelles, as it is shown in the figure [5,22,32,70,71]. Abbreviations: EE, early endosome; ER, endoplasmic reticulum; L, lysosome; LE/MVB, late endosome/ multivesicular body; RE, recycling endosome; TGN, trans- Golgi network. Rab7 Rab9 Rab24 LE/MVB Rab7 Rab4 EE Rab5 Rab5 Rab4 Rab5 Rab22 Rab11 RE Rab11 Rab17 Rab9 Rab11 Rab3A Secretory vesicles ER TGN Rab1 Rab2 Rab8 Rab8 Rab3B Rab13 Rab6 Rab8 Rab6 Rab12 Rab24 L Nucleus TRENDS in Endocrinology & Metabolism Figure I. enter a Rab4-mediated, rapid recycling pathway from early endosomes, which is similar to the rapid recycling pathway of TfRs. However, co-localization of b2ar, a rapidly recycling GPCR, with Rab11 has also been demonstrated suggesting that, similar to TfRs, these receptors can also recycle indirectly via recycling endosomes if they escape rapid recycling [24,25]. Recycling of GPCRs that remain associated with b-arrestins after endocytosis (Class B receptors, see below) is much slower [33]. These receptors proceed from sorting endosomes to slower recycling pathways or receptor degradation. The differential sensitivity of AT 1 Rrecycling pathways to wortmannin, a phosphatidylinositol (PtdIns) 3-kinase inhibitor, indicate that these slower recycling pathways are also heterogeneous [29]. Perinuclear recycling endosomes participate in the recycling of several GPCRs, including AT 1 R, V2 vasopressin receptor (V2R), m4achr and somatostatin receptors, which recycle through Rab11-dependent, slow pathway [31,34 36]. Degradation pathways During maturation the tubulo-vesicular sorting endosomes lose their tubular extensions, which contain

4 Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August β-arrestin? Early sorting endosome β-arrestin β-arrestin Ub Recycling endosome Late endosome/ multivesicular body Nucleus Lysosome EGFR GPCR Clathrin AP-2 TRENDS in Endocrinology & Metabolism Figure 2. Intracellular trafficking routes of plasma membrane receptors. EGFR and many GPCRs are endocytosed through clathrin-coated pits upon agonist activation. Some GPCRs can endocytose via non-coated vesicles indicated by question marks. Endocytotic vesicles fuse with each other and with early endosomes. Many agonists dissociate from their receptors in sorting endosomes. Individual GPCRs differ in the pattern of postendocyticsorting. Recycling can occur from early endosomes with rapid kinetics and this process is promoted by dissociation of b-arrestin (blue GPCR). Recyling can also occur from recycling endosomes (green GPCR) or MVB-like compartments (red GPCR) with much slower kinetics. Receptors targeted to degradation (black GPCR) follow the MVB or late endosome and lysosome pathway and are preferentially found in internal vesicles of MVB. Abbreviations: EGFR, epidermal growth factor receptor; GPCRs, G protein-coupled receptors; MVB, multivesicular body; Ub, ubiquitin. recycling receptors, and the bulky spherical endosomes translocate along microtubules toward the nucleus and become more acidic. This process leads to the formation of late endosomes, which do not receive transport vesicles directly from the plasma membrane, and are enriched in proteins targeted for degradation. Late endosomal elements are dynamic compartments with pleiomorphic organization, containing cisternal, tubular and vesicular regions with numerous membrane invaginations. Therefore they are frequently called multivesicular bodies (MVBs). Late endosomes are prelysosomal endocytic organelles, and their limiting membrane contains high amounts of LAMP1, a characteristic protein of lysosomes. Internal membranes in higher eukaryotic cells accumulate large amounts of lysobisphosphatidic acid, which has an inverted cone shape, and this was proposed to facilitate the formation of invaginations and internal vesicles of late endosomes [22,37,38]. Proteins destined to be degraded accumulate within MVB internal membranes, as first shown for the EGFR [39], leading to the idea that degradation is the fate of internal membranes in lysosomes. Fusion of the limiting membrane of the MVB with the lysosomal membrane results in the delivery of luminal vesicles and their contents to the hydrolytic interior of the lysosomes, where they are degraded. This mechanism enables degradation of transmembrane proteins, such as growth factor receptors or GPCRs. Membrane proteins that are excluded from these inner vesicles and remain in the limiting membrane of MVB can be recycled back to the plasma membrane or transported to other sites in the cell [37]. The boundary between late endosomes and lysosomes is elusive. Both compartments contain lysosomal enzymes, their ph is similarly acidic (ca 5.5), and their limiting membranes are primarily composed of the same lipids and proteins. Lysosomes can be identified based on their physical properties, on gradients and electron-dense appearance, and by the fact that they lack some proteins found on late endosomes, including phosphorylated hydrolase precursors, and Rab7 and Rab9 small GTPases [22,70]. Studies on regulation of certain GPCRs provided early evidence for the hypothesis that agonist-induced downregulation of total receptor pool is mediated by proteolysis in lysosomes [40]. Trafficking of the receptor to lysosomes has been imaged recently by fluorescence microscopy in living cells [41]. Lysosomal degradation of b2ar, d- and k-opioid receptors (DOR and KOR) has been demonstrated [42 44]. Inhibition of EGFR, KOR and

5 290 Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August 2004 CXC chemokine receptor 2 (CXCR2) degradation by dominant-negative Rab7, a small G protein required for lysosomal targeting, has also been shown [43,45,46], and overexpression of Rab7 targets AT 1 Rs toward lysosomes [31]. Endocytosis-independent proteolysis and non-lysosomal degradation of some GPCRs has also been suggested [41]. Agonist-induced non-lysosomal degradation of growth factor receptors [47] and opiate receptors [48] occur in proteasomes. However, proteasomes also degrade newly synthesized ubiquitylated DORs, which are retained during receptor synthesis [49]. Furthermore, non-endocytic degradation of b2ar is also insensitive to inhibitors of the proteasomes function [50], suggesting that alternative receptor degradation pathways do occur in some cells. Caveosomes Some plasma-membrane receptors enter cells through caveolae. There is limited knowledge about the trafficking routes of these vesicles, whether they are transported separately from cargoes endocytosed by clathrin-coated vesicles or whether they can fuse at some stages of intracellular trafficking. Recently, the existence of caveosomes has been suggested, but there is very little information about the functional relationship between caveosomes and endosomes [37,51]. Regulation of intracellular trafficking Segregation of the receptor from its cognate ligand is important during endosomal sorting, as small recycling vesicles have a high surface to volume ratio, which favors the recycling of receptors, whereas dissociated ligands are retained in bulky endosomes [21]. Although this geogeometric sorting mechanism can explain the targeting of dissociated ligands to lysosomes, differences in the recycling pathways and lysosomal targeting of plasma membrane receptors involves specific interactions with intracellular or membrane-associated proteins. Nutrient receptors are constitutively recycled back to the cell surface after dissociating from their cargo [4]. Because early studies did not identify the recycling signal, it was initially proposed that recycling to the cell surface occurs by default [22]. In accordance with this hypothesis, lysosomal targeting signals have been identified for several integral membrane proteins. These signals bear little similarity, suggesting that different sorting principles can operate simultaneously [4]. Targeting of tyrosine kinase receptors Lysosomal targeting of EGFR is a well-studied example for the operation of such sorting mechanisms. The vast majority of internalized EGFR is targeted to lysosomes by sorting signals. Cytoplasmic residues 945 to 991 of the EGFR contain a candidate tyrosine-based signal sequence ( 954 YLVI), which is similar to that found in other lysosome-targeted proteins, such as LAMP1 and LAMP2, and is a binding site for the sorting nexin (SNX1), which participates in lysosomal targeting of EGFR. SNX1 has also been shown to interact with a variety of other receptors, including the insulin receptor and PAR1, and forms a membrane coat complex with other proteins [4]. Two additional regions of EGFR have been shown to regulate lysosomal sorting of the receptor. Autophosphorylation of the tyrosine residue in the carboxyl terminal domain results in c-cbl binding through its SH2 domain and its phosphorylation. Phosphorylated c-cbl has ubiquitin ligase activity and also recruits ubiquitinactivating and conjugating enzymes to the receptor, and enhances its degradation in both proteasome- and lysosome-dependent ways [4,52,53]. Ubiquitin modification also regulates the down-regulation of the growth hormone receptor (GHR); however, in this case ubiquitylation of GHR itself is not required: rather, components of the down-regulation machinery are ubiquitylated in response to receptor activation [37,54]. A third lysosomal sorting signal contains a dileucine motif, and is located in the juxtamembrane region of EGFRs [4]. Targeting of GPCRs The role of the above mechanisms during the targeting of GPCRs has not been fully elucidated. Switching the sequence of the cytoplasmic tail of the lysosome-targeted PAR1 and the efficiently recycling substance P receptor reversed the targeting of these receptors, suggesting that interaction of sorting proteins with the cytoplasmic tail of the receptor is an important determinant of GPCR trafficking [55]. The C-terminal tail of GPCRs also has an important role in binding of b-arrestin molecules. The stability of a GPCR and b-arrestin binding following its internalization could dictate the intracellular trafficking fate of the receptor. Based on the stability of b-arrestinreceptor interaction, two functional classes of GPCRs were defined [56]. Class A receptors, including b2ar, ETA-R, MOR, preferentially bind b-arrestin2. However, this binding is weak; b-arrestin and the receptor dissociate shortly after endocytosis, and these receptors very rapidly and efficiently recycle back to the plasma membrane. In the case of class B receptors, such as AT 1 R, neurokinin 1 receptor, TRH receptor and V2R, which bind b-arrestin1 and b-arrestin2 with similar high affinity, b-arrestins bind tightly to the receptor and their colocalization can be detected in endosomes during the trafficking of the receptor [56 58]. The affinity of b-arrestin binding to the receptors depends on the presence of clusters containing serine and/or threonine residues localized within the C-terminal tail of these receptors [59], which were first identified as an internalization motif of AT 1 R [60]. In case of the b2ar, which lacks these residues, b-arrestin dissociation is required for dephosphorylation and resensitization of the receptor in endosomal compartments and facilitates its return to the plasma membrane. Class B receptors, including V2R and AT 1 R, bind b-arrestin tightly and recycle back to the cell surface slowly [33,59]. However, other studies indicate that long-term interaction between b-arrestin and V2R is not the only determinant of its intracellular retention [34]. Although recycling is considered the default pathway of receptor trafficking the number of signals that facilitate the recycling of GPCRs is rapidly increasing. The four amino acid sequence DSLL in the cytoplasmic tail of the b2ar receptor is required for rapid recycling. This C-terminal PDZ motif interacts with N-ethyl-maleimide

6 Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August sensitive factor (NSF) and/or Na C /H C exchanger regulatory factor/ezrin-radixin-moesin-binding phosphoprotein 50 (NHERF/EBP50), and interaction with NSF seems to play a crucial role in receptor recycling [61]. A different signal sequence essential for rapid recycling (LENLEAE) was recognized in the cytoplasmic tail of MOR. Fusion of this sequence with the C-terminal tail of the DOR changes its targeting from degradation to recycling [62]. A candidate for regulating the recycling of class B GPCRs is type 1 angiotensin II receptor-associated protein 1 (ARAP1), which has been shown to interact with the AT 1 R and enhance its recycling to the plasma membrane. However, the latter mechanism is not universal because ARAP1 has no effect on b2ar recycling [63]. Interestingly, b-arrestins are involved in the recycling of a chemoattractant receptor, which internalizes with a b-arrestin-independent mechanism [64]. The relevance of this mechanism to the recycling of hormone receptors, which internalize with the b-arrestinindependent mechanism, requires additional studies. Mechanisms involved in lysosomal targeting of GPCRs have also been described. GASP (GPCR-associated sorting protein) has been characterized as a regulator of postendocytic targeting of DOR, as its proper function is required for lysosomal sorting and proteolytic downregulation of DOR. By contrast, the other opioid receptor, MOR, bound only weakly to GASP, which is consistent with the differences in intracellular trafficking of the two receptors, because MOR can recycle efficiently [65]. Other GPCRs, such as b2ar, a2ar and the D4 dopamine receptor can also bind GASP; however, the functional importance of this interaction has not yet been determined [65]. The role of receptor ubiquitylation in lysosomal targeting was first identified in yeast, in which targeting of yeast mating factor receptors to vacuoles (organelles analogous to mammalian lysosomes) is dependent on the ubiquitylation of the receptor protein, because it targets these receptors to internal vesicles of the MVB [6]. Ubiquitin also has a role in mammalian cells in the posttranslational sorting of the G protein-coupled CXCR4. Mutation of ubiquitin-acceptor lysine residues in a degradation motif (SSLKILSKGK) situated in the cytoplasmic tail of the receptor did not affect CXCR4 internalization, but its degradation was completely inhibited [66]. In a very recent study E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) has been shown to mediate ubiquitylation and sorting of CXCR4 [67]. Ubiquitylation of the b2ar has also been shown to be required for its down-regulation [8]. Similar results were obtained with the V2R because receptor stimulation leads to rapid b-arrestin-dependent ubiquitylation and increased degradation [68]. By contrast, DOR trafficking to lysosomes seems to be independent of ubiquitylation, suggesting that this process is also not universal [9]. Ubiquitin modification of b-arrestin after agonist treatment has also been published; this process seems to be important for internalization of the receptor [8]. In addition, recent studies on b2ar and V2R suggest that the time course of ubiquitylation and deubiquitylation of b-arrestin correlates with association dissociation kinetics of b-arrestin-receptor binding. b-arrestin ubiquitin chimera stably associates with b2ar, and therefore results in enhancement of its internalization and degradation [65,69]. Thus, these studies argue that ubiquitylation is important for the lysosomal targeting and down-regulation of some hormone receptors. Furthermore, ubiquitin not only serves as a sorting tag on proteins, but also can act as a regulator of the trafficking machinery [37]. Concluding remarks and future perspectives Although the major intracellular trafficking pathways of plasma membrane receptors have been mapped, owing to the inherent complexity of the endosomal system, our understanding of these pathways and the signals that target molecules to different pathways is still incomplete. The diversity of intracellular trafficking routes also complicates the identification of the signals responsible for intracellular targeting of the receptors, and more studies are required to elucidate the exact mechanisms of endosomal targeting. 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