Intracellular trafficking of hormone receptors

Intracellular trafficking of hormone receptors

Review TRENDS in Endocrinology and Metabolism Vol.15 No.6 August 2004 Intracellular trafficking of hormone receptors Zsuzsanna Ga´borik and La´szlo...

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Review

TRENDS in Endocrinology and Metabolism

Vol.15 No.6 August 2004

Intracellular trafficking of hormone receptors Zsuzsanna Ga´borik and La´szlo´ 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. AT1 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: La´szlo´ Hunyady ([email protected]). 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 AT1 angiotensin receptor (AT1R) 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].

www.sciencedirect.com 1043-2760/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2004.06.009

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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].

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 www.sciencedirect.com

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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), b2 adrenergic receptor (b2AR), D2R] [24,26,28]. Although Rab5 directly interacts with the C-terminal tail of the AT1R, 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 AT1R [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

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Box 1. Molecular architecture of intracellular organelles involved in membrane trafficking 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.

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

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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 AT1R recycling www.sciencedirect.com

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 AT1R, 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

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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 postendocytic sorting. 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, www.sciencedirect.com

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, dand k-opioid receptors (DOR and KOR) has been demonstrated [42–44]. Inhibition of EGFR, KOR and

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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 AT1Rs 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 (954YLVI), 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]. www.sciencedirect.com

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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 1022–1063 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 AT1R, 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 AT1R [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 AT1R, 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

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sensitive factor (NSF) and/or NaC/HC 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 AT1R 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 www.sciencedirect.com

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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. New technical advances, such as the use of fluorescent reporters (e.g. green fluorescent proteintagged molecules), progress in real-time imaging, applications of biophysical methods to test protein–protein interactions (e.g. using resonance energy transfer) and new methods for disrupting the function of proteins (e.g. knock-out animal models or the use of small interfering RNAs) will lead to a more complete understanding of these mechanisms. Identification of the intracellular trafficking pathways will facilitate the understanding of the role of these compartments in signaling and regulation of hormone receptors. Acknowledgements This work was supported in part by a Collaborative Research Initiative Grant from the Wellcome Trust (069416/Z/02/Z), and by grants from the Hungarian Ministry of Public Health (ETT 036/2003) and the Hungarian Science Foundation (OTKA T-046445 and OTKA Ts-040865).

References 1 Lefkowitz, R.J. (1998) G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J. Biol. Chem. 273, 18677–18680 2 Sorkin, A. and Von Zastrow, M. (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat. Rev. Mol. Cell Biol. 3, 600–614 3 Hunyady, L. et al. (2000) Mechanisms and functions of AT(1) angiotensin receptor internalization. Regul. Pept. 91, 29–44 4 Kurten, R.C. (2003) Sorting motifs in receptor trafficking. Adv. Drug Deliv. Rev. 55, 1405–1419 5 Maxfield, F.R. and McGraw, T.E. (2004) Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132 6 Hicke, L. and Dunn, R. (2003) Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172 7 Mihalik, B. et al. (2003) Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threoninerich region. Int. J. Biochem. Cell Biol. 35, 992–1002 8 Shenoy, S.K. et al. (2001) Regulation of receptor fate by ubiquitination of activated beta 2- adrenergic receptor and beta-arrestin. Science 294, 1307–1313 9 Tanowitz, M. and Von Zastrow, M. (2002) Ubiquitination-independent trafficking of G protein-coupled receptors to lysosomes. J. Biol. Chem. 277, 50219–50222 10 Ferguson, S.S.G. (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53, 1–24 11 Thomas, W.G. and Qian, H. (2003) Arresting angiotensin type 1 receptors. Trends Endocrinol. Metab. 14, 130–136

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12 Conner, S.D. and Schmid, S.L. (2003) Regulated portals of entry into the cell. Nature 422, 37–44 13 Johannes, L. and Lamaze, C. (2002) Clathrin-dependent or not: is it still the question?. Traffic 3, 443–451 14 Roettger, B.F. et al. (1995) Dual pathways of internalization of the cholecystokinin receptor. J. Cell Biol. 128, 1029–1041 15 Vickery, R.G. and Von Zastrow, M. (1999) Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes. J. Cell Biol. 144, 31–43 16 Okamoto, Y. et al. (2000) Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells. J. Biol. Chem. 275, 6439–6446 17 Rapacciuolo, A. et al. (2003) Protein kinase A and G proteincoupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways. J. Biol. Chem. 278, 35403–35411 18 Ga´borik, Z. et al. (2001) b-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor. Mol. Pharmacol. 59, 239–247 19 Zhang, J. et al. (1996) Dynamin and b-arrestin reveal distinct mechanisms for G protein- coupled receptor internalization. J. Biol. Chem. 271, 18302–18305 20 Di Guglielmo, G.M. et al. (2003) Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover. Nat. Cell Biol. 5, 410–421 21 Mellman, I. (1996) Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625 22 Gruenberg, J. (2001) The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol. 2, 721–730 23 Miller, K. et al. (1986) Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells. J. Cell Biol. 102, 500–509 24 Seachrist, J.L. et al. (2000) beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases. J. Biol. Chem. 275, 27221–27228 25 Moore, R.H. et al. (1995) Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes. J. Cell Sci. 108, 2983–2991 26 Volpicelli, L.A. et al. (2001) Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies. J. Biol. Chem. 276, 47590–47598 27 Bremnes, T. et al. (2000) Regulation and Intracellular Trafficking Pathways of the Endothelin Receptors. J. Biol. Chem. 275, 17596–17604 28 Iwata, K. et al. (1999) Dynamin and rab5 regulate GRK2-dependent internalization of dopamine D2 receptors. Eur. J. Biochem. 263, 596–602 29 Hunyady, L. et al. (2002) Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. J. Cell Biol. 157, 1211–1222 30 Seachrist, J.L. et al. (2002) Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion. J. Biol. Chem. 277, 679–685 31 Dale, L.B. et al. (2004) Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases. J. Biol. Chem. 279, 13110–13118 32 Rosenfeld, J.L. et al. (2002) Regulation of G-protein-coupled receptor activity by rab GTPases. Receptors Channels 8, 87–97 33 Oakley, R.H. et al. (1999) Association of b-arrestin with G proteincoupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J. Biol. Chem. 274, 32248–32257 34 Innamorati, G. et al. (2001) The long and the short cycle. Alternative intracellular routes for trafficking of G-protein-coupled receptors. J. Biol. Chem. 276, 13096–13103 35 Kreuzer, O.J. et al. (2001) Agonist-mediated endocytosis of rat somatostatin receptor subtype 3 involves beta-arrestin and clathrin coated vesicles. J. Neuroendocrinol. 13, 279–287 36 Volpicelli, L.A. et al. (2002) Rab11a and myosin Vb regulate recycling of the M4 muscarinic acetylcholine receptor. J. Neurosci. 22, 9776–9784 37 Katzmann, D.J. et al. (2002) Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3, 893–905 38 Kobayashi, T. et al. (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193–197 www.sciencedirect.com

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39 Felder, S. et al. (1990) Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell 61, 623–634 40 Von Zastrow, M. (2001) Role of endocytosis in signalling and regulation of G-protein-coupled receptors. Biochem. Soc. Trans. 29, 500–504 41 Tsao, P. et al. (2001) Role of endocytosis in mediating downregulation of G-protein-coupled receptors. Trends Pharmacol. Sci. 22, 91–96 42 Tsao, P.I. and Von Zastrow, M. (2000) Type-specific sorting of G protein-coupled receptors after endocytosis. J. Biol. Chem. 275, 11130–11140 43 Li, J.G. et al. (2000) Mechanisms of agonist-induced down-regulation of the human kappa- opioid receptor: internalization is required for down-regulation. Mol. Pharmacol. 58, 795–801 44 Moore, R.H. et al. (1999) Agonist-induced sorting of human beta2adrenergic receptors to lysosomes during downregulation. J. Cell Sci. 112, 329–338 45 McCaffrey, M.W. et al. (2001) Rab4 affects both recycling and degradative endosomal trafficking. FEBS Lett. 495, 21–30 46 Fan, G.H. et al. (2003) Differential regulation of CXCR2 trafficking by Rab GTPases. Blood 101, 2115–2124 47 Strous, G.J. and Govers, R. (1999) The ubiquitin-proteasome system and endocytosis. J. Cell Sci. 112, 1417–1423 48 Chaturvedi, K. et al. (2001) Proteasome involvement in agonistinduced down-regulation of mu and delta opioid receptors. J. Biol. Chem. 276, 12345–12355 49 Petaja-Repo, U.E. et al. (2001) Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome. J. Biol. Chem. 276, 4416–4423 50 Jockers, R. et al. (1999) Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis. J. Biol. Chem. 274, 28900–28908 51 Pelkmans, L. et al. (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat. Cell Biol. 3, 473–483 52 Joazeiro, C.A. et al. (1999) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312 53 Levkowitz, G. et al. (1998) c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12, 3663–3674 54 Govers, R. et al. (1999) Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor. EMBO J. 18, 28–36 55 Trejo, J. and Coughlin, S.R. (1999) The cytoplasmic tails of proteaseactivated receptor-1 and substance P receptor specify sorting to lysosomes versus recycling. J. Biol. Chem. 274, 2216–2224 56 Oakley, R.H. et al. (2000) Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275, 17201–17210 57 Anborgh, P.H. et al. (2000) Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors. Mol. Endocrinol. 14, 2040–2053 58 Claing, A. et al. (2002) Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and b-arrestin proteins. Prog. Neurobiol. 66, 61–79 59 Oakley, R.H. et al. (2001) Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-betaarrestin complexes after receptor endocytosis. J. Biol. Chem. 276, 19452–19460 60 Hunyady, L. et al. (1994) Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of the AT1 angiotensin receptor. J. Biol. Chem. 269, 31378–31382 61 Xiang, Y. and Kobilka, B. (2003) The PDZ-binding motif of the beta2adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes. Proc. Natl. Acad. Sci. U. S. A. 100, 10776–10781 62 Tanowitz, M. and Von Zastrow, M. (2003) A novel endocytic recycling signal that distinguishes the membrane trafficking of naturally occurring opioid receptors. J. Biol. Chem. 278, 45978–45986 63 Guo, D.F. et al. (2003) Type I angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane. Biochem. Biophys. Res. Commun. 310, 1254–1265

Review

TRENDS in Endocrinology and Metabolism

64 Vines, C.M. et al. (2003) N-formyl peptide receptors internalize but do not recycle in the absence of arrestins. J. Biol. Chem. 278, 41581–41584 65 Whistler, J.L. et al. (2002) Modulation of postendocytic sorting of G protein-coupled receptors. Science 297, 615–620 66 Marchese, A. and Benovic, J.L. (2001) Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. 276, 45509–45512 67 Marchese, A. et al. (2003) The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Dev. Cell 5, 709–722

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68 Martin, N.P. et al. (2003) Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination. J. Biol. Chem. 278, 45954–45959 69 Shenoy, S.K. and Lefkowitz, R.J. (2003) Trafficking patterns of betaarrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination. J. Biol. Chem. 278, 14498–14506 70 Somsel Rodman, J. and Wandinger-Ness, A. (2000) Rab GTPases coordinate endocytosis. J. Cell Sci. 113, 183–192 71 Takai, Y. et al. (2001) Small GTP-binding proteins. Physiol. Rev. 81, 153–208

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