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β-arrestins: traffic cops of cell signaling
b-arrestins: traffic cops of cell signaling Robert J Lefkowitz1 and Erin J Whalen2 Once thought to function only in the desensitization of seven membrane spanning receptors (7MSRs), the ubiquitous barrestin molecules are increasingly appreciated to play important roles in the endocytosis and signaling of these receptors. These functions reflect the ability of the b-arrestins to bind an evergrowing list of signaling and endocytic elements, often in an agonist-dependent fashion. One heavily studied system is that leading to MAP kinase activation via b-arrestin-mediated scaffolding of these pathways in a receptor-dependent fashion. The b-arrestins are also found to be involved in the regulation of novel receptor systems, such as Frizzled and TGFb receptors. Addresses 1 Howard Hughes Medical Institute, Duke University Medical Center, DUMC Box 3821, Durham, NC 27710, USA e-mail: [email protected] 2 Duke University Medical Center, DUMC Box 3821, Durham, NC 27710, USA e-mail: [email protected]
Current Opinion in Cell Biology 2004, 16:162–168 This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes 0955-0674/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2004.01.001
Abbreviations 7MSR seven-membrane-spanning receptor b2AR b2-adrenergic receptor GRK G-protein-coupled receptor kinases Ral-GDS Ral GDP dissociation stimulator
Introduction The arrestins constitute a small gene family with four members, all of which interact with seven-membranespanning receptors (7MSRs) after these receptors have been activated and phosphorylated by G-protein-coupled receptor kinases (GRKs) [1,2], (Figure 1). Arrestin1 and arrestin4 (x-arrestin) are found exclusively in retinal rods and cones, respectively, where they regulate rhodopsin and color opsins. By contrast, arrestin2 and -3 (most often referred to as b-arrestin1 and b-arrestin2) are expressed in virtually all tissues, where they regulate most 7MSRs. Originally discovered in the context of desensitization of rhodopsin [3] and the b2-adrenergic receptor (b2AR) [4–6], it has recently been increasingly appreciated that the two forms of b-arrestin also serve as multi-functional adaptors that link the receptors to the endocytic machinery associated with the formation of clathrin-coated pits Current Opinion in Cell Biology 2004, 16:162–168
[7,8], (Figure 1c), as well as to an ever-growing list of signaling molecules [9,10]. This article summarizes advances over the past two years in understanding these novel adaptor functions of the b-arrestins.
Desensitization The paradigmatic function of the b-arrestins is their ability to desensitize 7MSRs by sterically blocking their interaction with heterotrimeric G proteins (Figure 1b). In the case of the b2AR, this mechanism was originally documented in a reconstituted system of purified recombinant proteins [4–6]. However, recently Perry et al. [9] demonstrated that the b-arrestins bind cAMP phosphodiesterases of the PDE4D family. Upon activation of the b2AR, b-arrestin2 recruits PDE4D isoforms into a complex with the activated receptor, where it is positioned to degrade cAMP at an enhanced rate. This slows the rate of PKA activation. Thus, b-arrestin not only slows the rate of b2AR/Gsstimulated cAMP generation, but in a coordinated fashion increases the rate of cAMP degradation in proximity to the receptor–adenylate-cyclase signaling unit.
Receptor trafficking Endocytosis of activated 7MSRs is a fairly general phenomenon. It may be mediated by clathrin-coated pits, caveolae or other uncoated vesicles [7,8]. Once internalized, receptors may be dephosphorylated, resensitized and recycled to the cell surface, be targeted to lysosomes for degradation, or engage in additional intracellular signaling. b-arrestins-1 and -2 play a central role in mediating the clathrin-dependent internalization of 7MSRs by serving as adaptors linking the receptors to elements of the endocytotic machinery. These endocytic elements include clathrin [10], the clathrin adaptor AP2 [11], the small G protein ARF6 [12] and its guanine nucleotide exchange factor, ARNO, and NSF (N-ethylmelaimide sensitive fusion protein) [13] (Figure 1c). Quite recently, the spectrum of receptors that can utilize b-arrestins for clathrin-mediated endocytosis has significantly expanded. b-arrestin2 was shown to be required for internalization of the 7MSR Frizzled-4 after the receptor’s stimulation by the ligand Wnt5A [14]. In contrast to virtually all previously studied examples, where barrestins are recruited directly to a GRK-phosphorylated receptor, in this case b-arrestin binds to PKC-phosphorylated Dvl2, an adaptor protein previously shown to interact with Fz to mediate its canonical signaling through b-catenin (Figure 2a). Perhaps even more surprisingly, one of the receptors for TGF-b, the single-membrane-spanning TGF-b type-III www.sciencedirect.com
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Figure 1
(a) Coupling to G protein
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Classical model of (a) 7MSR activation and signaling, (b) GRK-mediated receptor phosphorylation and b-arrestin mediated desensitization, and (c) b-arrestin-mediated clathrin/AP-2 dependent receptor endocytosis.
receptor, is also internalized by a b-arrestin2-dependent process [15]. It is the only one of more than a dozen different types of TGF-b receptors found to do so when tested. Interestingly, the interaction of b-arrestin2 with the cytoplasmic tail of TbRIII also requires its phosphorylation. However, this is catalyzed by the TGF-b type-II receptor, which is itself a S/T kinase, rather than by a GRK. TbRII and TbRIII then internalize together (Figure 2b). Ubiquitination appears to be centrally involved in barrestin-mediated endocytosis of 7MSRs. b-arrestins bind MDM2 [16,17], an E3 ubiquitin ligase best known for its role in regulating the tumor suppressor p53. When a receptor such as b2AR or the vasopressin receptor is stimulated, it binds b-arrestin, triggering MDM2mediated ubiquitination of the b-arrestin [16,18]. This ubiquitination step is required for b-arrestin to perform its adaptor role in clathrin-mediated endocytosis, although the mechanisms responsible for this have not been elucidated. Interestingly, b-arrestins also act as obligatory adaptors to bring E3 ligases to the receptors, which are also ubiquitinated in a stimulus-dependent fashion [16]. At least in the case of the b2AR, however, this ligase appears not to be MDM2. In contrast with the situation originally described for the yeast STE2 receptor [19], www.sciencedirect.com
receptor ubiquitination is not required for the endocytosis of mammalian 7MSRs. Rather, as shown for the b2AR [16], CXCR4 receptor [20], V2 vasopressin receptor [21] and PAF [22], receptor ubiquitination is necessary for post-endosomal sorting to lysosomes. There are interesting and mechanistically significant differences in the patterns of 7MSR trafficking related to how the receptors interact with b-arrestins. As originally described by Oakley et al., two distinct patterns can be observed [23]. So called ‘class A’ receptors, such as the b2AR, bind b-arrestin transiently, traffic with it to clathrin-coated pits and then dissociate. The receptors then internalize without b-arrestin, and generally recycle relatively rapidly. By contrast, ‘class B’ receptors, such as the AT1A angiotensin II receptor or the V2 vasopressin receptor, bind b-arrestin more tightly and internalize together with it. Such receptors recycle more slowly. Class A receptors tend to bind b-arrestin2 preferentially whereas Class B receptors generally have equal affinity for both forms of b-arrestin [23]. These distinct patterns appear to reflect differing rates of deubiquitination of activated receptor-bound b-arrestin, a process which appears to determine the dissociation of the b-arrestin from the receptors [18]. Class B but not Class A receptors provoke sustained b-arrestin ubiquitination. In fact, when Current Opinion in Cell Biology 2004, 16:162–168
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Figure 2
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b-arrestin-mediated endocytosis of the Frizzled 4 receptor and non-7MSRs. (a) b-arrestin2-mediated endocytosis of the Wnt5a-stimulated Frizzled 4 (Fz4) receptor mediated by protein kinase C (PLC) phosphorylation of the intracellular b-arrestin adaptor Dishevelled 2 (Dvl2). (b) b-arrestin2-mediated internalization of TGF-b1 receptor subtypes RII and RIII, facilitated by RII phosphorylation of T841 on RIII. (c) b-arrestin-mediated internalization of the IGF1 receptor.
a chimeric molecule consisting of b-arrestin2 with ubiquitin fused in frame to the C terminus, which cannot be deubiquitinated, is transfected into cells, it converts the pattern of endocytosis of class A receptors to that of class B receptors [18]. Perhaps because of their ability to bind b-arrestins more tightly, class B receptors can functionally sequester pools of b-arrestin, thus leading to heterologous regulation of the endocytosis of other receptors. Thus, activation of the NK1 neurokinin receptor inhibits endocytosis of the NK3 receptor [24], and activation of the V2 vasopressin receptor inhibits internalization of the b2AR [25].
Figure 3
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Signaling The most rapidly expanding area of research on the barrestins over the past few years relates to their multifaceted roles as signaling adaptors and scaffolds connecting 7MSRs with an ever-growing list of effector pathways [2,26]. This was initially appreciated in relation to Src family non-receptor tyrosine kinases [27]. Recently, most attention has been directed toward the MAP kinases (Figure 3). These enzymes are downstream elements of highly conserved cascades of MAPKKKs and MAPKKs leading to activation of the MAP kinases, such as the ERKs, JNKs and p38 [28]. Classically, activated MAPKs translocate to the nucleus where they phosphorylate and activate transcription factors. However, a growing list of
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New paradigm for 7MSR signaling, including (a) heterotrimeric-Gprotein-dependent and (b) b-arrestin-dependent mechanisms. There also exists the potential for signaling that is dependent on both heterotrimeric G protein and b-arrestin. www.sciencedirect.com
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cytosolic substrates for the MAPKs has been identified in recent years. The heterogeneity and diversity of kinases at each level of the MAPK cascades (for example, there are five ERKs, four p38s and three JNKs) suggested that scaffolding molecules might be used to ensure fidelity and efficiency in activating specific MAPK modules. In the 1990s several such molecules were found, initially in yeast [29] and then in mammalian systems [30]. Recently, it has been found that b-arrestin2 also functions as a scaffold for 7MSR activation of several mammalian MAPKs including ERK1/2 [31–33], JNK3 [34] and at least one of the p38s [35], (Figure 3b). Not only does the b-arrestin form complexes with individual members of a particular MAPK cassette, thereby facilitating activation of, for example, JNK3 by ASK or ERK1/2 by Raf, but it retains the activated MAPK in the cytoplasm, thereby presumably directing phosphorylation of specific cytoplasmic substrates [31–34], (Figure 3b). Coordinately, it inhibits phosphorylation of nuclear transcription factors, thereby actually inhibiting ERK-dependent transcription [36,37]. This ability of b-arrestin2 to retain activated scaffolded MAPKs in the cytoplasm is apparently related to the presence of a leucine-rich nuclear export signal in the C terminus of b-arrestin2, but not of b-arrestin1. This motif appears to mediate nuclear export of b-arrestin2 by a leptomycin-B-sensitive process [38,39]. 7MSR-stimulated b-arrestin-mediated scaffolding and activation of MAPKs appears to be intimately linked to endocytosis of the receptor–b-arrestin complex, although the molecular details of this linkage remain obscure. Class B 7MSRs bind b-arrestins tightly and internalize with them, and are thus much more effective activators of such b-arrestin-scaffolded MAPK pathways [36,37]. In fact, simply switching the C termini between the b2AR (class A) and the V2R (class B) receptors by construction of chimeric molecules is sufficient to change their patterns of b-arrestin binding [40], trafficking [18,40] and MAPK activation [37]. A major question concerning the role of b-arrestins in mediating signaling by 7MSRs is whether they operate in series or in parallel with the classical signal mediator, the heterotrimeric G proteins. Recently, it was demonstrated, at least in the case of the Gq-coupled AT1A angiotensin receptor, that b-arrestin2 can mediate signaling to ERK completely independently of G-protein activation (Figure 3b). This was established using a receptor mutant (DRY AAY) as well as a mutated angiotensin peptide (SII) [41]. Neither the receptor, when activated by Ang II, nor the peptide, when used to activate the WT receptor, are able to activate G proteins. Nonetheless, both stimulate ERK activation and recruit b-arrestin2. The ability of the mutant receptor and of the mutant peptide to activate ERK is completely ablated by treatment of cells with siRNA, which reduces expression of b-arrestin2. Interestingly, 50% of WT–Ang stimulation of ERK www.sciencedirect.com
through the WT receptor is eliminated by the application of siRNA to b-arrestin2, whereas the remaining 50% is blocked by a PKC inhibitor [41]. These findings suggest that two types of pathways mediate ERK activation in response to Ang stimulation, one mediated by G proteins, the other by b-arrestin2 (Figure 3). The results, however, in no way exclude the possibility that in other systems G proteins and b-arrestins might operate sequentially to activate effectors. Another example of apparently Gprotein-independent, b-arrestin-dependent activation of ERK has been described by Azzi et al. [42] It was found that, for both the b2AR and the V2 vasopressin receptor, inverse agonists for Gs-activated adenylate cyclase are in fact positive partial agonists for b-arrestin-dependent ERK activation. Findings such as these also reinforce the idea that there may be multiple ‘active’ conformations of a receptor [43]. In these cases, it would appear that distinct conformations of the receptor are able to activate different types of effectors such as G proteins and b-arrestin2. Such results have significant and obvious implications for the development of novel therapeutic agents. Several examples have also recently been published of b-arrestin-dependent PtdIns-3-kinase/AKT activation. Thrombin, which acts through a 7MSR, stimulates AKT through b-arrestin1-dependent and -independent pathways, with distinct time courses and physiological consequences [44]. The IGF1 receptor, which is itself a tyrosine kinase, was also shown to activate the PtdIns-3kinase/AKT system via a b-arrestin1-dependent process, leading to anti-apoptotic effects [45]. This pathway is independent of the receptor’s tyrosine kinase activity. The molecular details of these signaling pathways remain to be determined. Recently, an interesting example of heterologous regulation of b-arrestin-mediated ERK activation has been presented. Olefsky and colleagues demonstrated that insulin stimulation of rat1 fibroblasts leads to phosphorylation (S412) and ubiquitination of b-arrestin1 [46,47]. After short periods of stimulation, the b-arrestin phosphorylation functionally impairs ERK activation by IGF1, LPA and isoproterenol [46], whereas after longer periods of stimulation ubiquitination of b-arrestin leads to its downregulation, which also impairs ERK activation through a variety of receptors [47].
Chemotaxis Several recent studies indicate an as yet poorly defined role or roles for b-arrestin2 in chemotaxis to a variety of chemoattractants. Splenic lymphocytes (T and B cells) from b-arrestin2 knockout mice show strikingly impaired chemotaxis in vitro to SDF, which is mediated by the CXCR4 receptor [48]. This has also been demonstrated for CXCR4 receptors transfected into HEK293 cells, Current Opinion in Cell Biology 2004, 16:162–168
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where it was further shown that b-arrestin2-dependent activation of p38 MAPK is required for the chemotactic response [35]. The potential pathophysiological significance of these observations has been demonstrated in a mouse model of allergic asthma (ovalbumin sensitization) which does not develop in b-arrestin2 knockout mice, apparently because of a failure of T lymphocytes to accumulate in their airways [49]. Recent findings of Ge et al. suggest that the role of barrestins in chemotaxis may be related to their mediation of MAP kinase activation as described above [50]. Activation of protease-activated receptor-2 (PAR-2) in NIH3T3 cells promotes b-arrestin- and ERK1/2dependent reorganization of the actin cytoskeleton, extension of polarized pseudopodia and chemotaxis. Further, PAR2/b-arrestin/ERK scaffolding complexes are enriched in the pseudopodia [50]. Another mechanism by which b-arrestins may participate in cytoskeletal reorganization during chemotaxis is by binding and translocating Ral GDP dissociation stimulator (Ral-GDS, which promotes GDP dissociation from the small G protein Ral), thereby leading to activation of the Ral effector pathway [51].
Conclusions The b-arrestins have recently emerged as important adaptors and scaffolds linking the activated forms of 7MSRs to a rapidly growing list of cellular signaling systems. They also mediate the clathrin-coated pit internalization of many receptors via their interactions with several components of the endocytic machinery, a process regulated by the ubiquitination and deubiquitination of the b-arrestins. The most thoroughly studied b-arrestindependent signaling system is that leading to ERK activation. The mechanisms by which the b-arrestins scaffold such MAP kinase pathways may be intimately linked to their roles in receptor endocytosis. Physiologic consequences of such b-arrestin dependent, G-proteinindependent signaling are likely to differ from those of G-protein-mediated activation of the same effectors. Delineation of the physiological consequences of barrestin-mediated signaling, as well as of the structural basis for this multi-faceted regulation, is likely in the next few years, as is a further significant expansion of the range of systems in which b-arrestins play important roles.
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MAPK scaffold for the activation of JNK3. Science 2000, 290:1574-1577. 35. Sun Y, Cheng Z, Ma L, Pei G: b-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol Chem 2002, 277:49212-49219. These findings clearly support the role of b-arrestins as signaling molecules involved in the complex process of CXCR4-stimulated chemotaxis. 36. Tohgo A, Pierce KL, Choy EW, Lefkowitz RJ, Luttrell LM: b-arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. J Biol Chem 2002, 277:9429-9436. 37. Tohgo A, Choy EW, Gesty-Palmer D, Pierce KL, Laporte S, Oakley RH, Caron MG, Lefkowitz RJ, Luttrell LM: The stability of the G-protein-coupled-receptor–b-arrestin interaction determines the mechanism and functional consequence of ERK activation. J Biol Chem 2003, 278:6258-6267. 38. Scott MG, Le Rouzic E, Perianin A, Pierotti V, Enslen H, Benichou S, Marullo S, Benmerah A: Differential nucleocytoplasmic shuttling of b-arrestins. Characterization of a leucine-rich nuclear export signal in b-arrestin2. J Biol Chem 2002, 277:37693-37701. Scott et al. describe the nucleocytoplasmic shuttling of b-arrestin2, the cytoplasmic localization of which is due to a c-terminal nuclear export sequence. Mutating the nuclear export sequence leads to the accumulation of b-arrestin2 in the nucleus. These studies further demonstrate the co-localization of JNK3 and b-arrestin2 within the cell. 39. Wang P, Wu Y, Ge X, Ma L, Pei G: Subcellular localization of b-arrestins is determined by their intact N domain and the nuclear export signal at the C terminus. J Biol Chem 2003, 278:11648-11653. 40. Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG: Molecular determinants underlying the formation of stable intracellular G-protein-coupled-receptor–b-arrestin complexes after receptor endocytosis. J Biol Chem 2001, 276:19452-19460. 41. Wei H, Ahn S, Shenoy SK, Karnik SS, Hunyady L, Luttrell LM, Lefkowitz RJ: Independent b-arrestin2 and G-protein-mediated pathways for angiotensin II activation of extracellular signalregulated kinases 1 and 2. Proc Natl Acad Sci U S A 2003, 100:10782-10787. These studies are the first to demonstrate 7MSR-stimulated b-arresin-2mediated Erk activation, completely independent of heterotrimeric Gprotein activation. 42. Azzi M, Charest PG, Angers S, Rousseau G, Kohout T, Bouvier M, Pineyro G: b-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G-proteincoupled receptors. Proc Natl Acad Sci U S A 2003, in press. ICI118551 and propranolol, inverse agonists for b2AR-stimulated adenylate cyclase, stimulated b2AR-mediated activation of ERK1/2 independently of Gs/Gi. These studies demonstrate b-arrestin-dependent, heterotrimeric-G-protein-independent b2AR signaling, which suggests ligand-specific receptor conformations and intracellular signaling pathways. 43. Kenakin T: Ligand-selective receptor conformations revisited: the promise and the problem. Trends Pharmacol Sci 2003, 24:346-354. 44. Goel R, Phillips-Mason PJ, Raben DM, Baldassare JJ: a-thrombin induces rapid and sustained Akt phosphorylation by barrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression. J Biol Chem 2002, 277:18640-18648. 45. Povsic T, Kohout TA, Lefkowitz RJ: b-arrestin1 mediates IGF-1 activation of PI-3-K and anti-apoptosis. J Biol Chem 2003, in press. 46. Dalle S, Imamura T, Rose DW, Worrall DS, Ugi S, Hupfeld CJ, Olefsky JM: Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating b-arrestin1. Mol Cell Biol 2002, 22:6272-6285. 47. Hupfeld CJ, Dalle S, Olefsky JM: b-arrestin 1 down-regulation after insulin treatment is associated with supersensitization of b2 adrenergic receptor Gas signaling in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A 2003, 100:161-166. Current Opinion in Cell Biology 2004, 16:162–168
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Stimulation with insulin down-regulates b-arrestin1 leading to an increase in b2AR-stimulated cAMP generation. These studies reveal a mechanism by which insulin receptor stimulation can regulate 7MSR function/regulation by affecting b-arrestin1-mediated receptor desensitization and internalization. 48. Fong AM, Premont RT, Richardson RM, Yu YR, Lefkowitz RJ, Patel DD: Defective lymphocyte chemotaxis in b-arrestin2- and GRK6-deficient mice. Proc Natl Acad Sci U S A 2002, 99:7478-7483. Differential regulation and signaling involved in the complex processes driving CXCL12 chemotaxis by different GRKs and b-arrestin2. These studies demonstrate a cell-type-specific chemotactic defect and a positive regulatory role for GRK6 and b-arrestin2 in mediating the chemotactic responses of T and B lymphocytes. 49. Walker J, Fong A, Lawson B, Savov J, Patel D, Schwartz D, Lefkowitz R: b-arrestin2 regulates the development of allergic asthma. J Clin Invest 2003, in press. b-arrestin2-deficient mice are resistant to the development of allergic asthma. These studies implicate b-arrestin2 in the mediation of immune components thought to play a key role in the development of allergic
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asthma. A deficit in b-arrestin2-dependent T-cell chemotaxis to the lung may underlie the phenotype. 50. Ge L, Ly Y, Hollenberg M, DeFea K: A b-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis. J Biol Chem 2003, 278:34418-34426. The authors demonstrate increased b-arrestin-associated phosphoERK1/2 in pseudopodia during PAR2-induced chemotaxis, suggesting a physiological role for b-arrestin-mediated scaffolding in chemotaxis. 51. Bhattacharya M, Anborgh PH, Babwah AV, Dale LB, Dobransky T, Benovic JL, Feldman RD, Verdi JM, Rylett RJ, Ferguson SS: b-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization. Nat Cell Biol 2002, 4:547-555. b-arrestins bind inactive Ral-GDS in the cytosol. fMLP-receptor stimulation leads to the dissociation of this complex and translocation of active Ral-GDS and b-arrestin to the plasma membrane. Re-association of this complex leads to Ral-GDS inactivation. These studies demonstrate 7MSR-mediated regulation of the Ras GTPase, Ral, by b-arrestin.
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Current Opinion in Cell Biology 2004, 16:162–168 This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes 0955-0674/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2004.01.001
Abbreviations 7MSR seven-membrane-spanning receptor b2AR b2-adrenergic receptor GRK G-protein-coupled receptor kinases Ral-GDS Ral GDP dissociation stimulator
Introduction The arrestins constitute a small gene family with four members, all of which interact with seven-membranespanning receptors (7MSRs) after these receptors have been activated and phosphorylated by G-protein-coupled receptor kinases (GRKs) [1,2], (Figure 1). Arrestin1 and arrestin4 (x-arrestin) are found exclusively in retinal rods and cones, respectively, where they regulate rhodopsin and color opsins. By contrast, arrestin2 and -3 (most often referred to as b-arrestin1 and b-arrestin2) are expressed in virtually all tissues, where they regulate most 7MSRs. Originally discovered in the context of desensitization of rhodopsin [3] and the b2-adrenergic receptor (b2AR) [4–6], it has recently been increasingly appreciated that the two forms of b-arrestin also serve as multi-functional adaptors that link the receptors to the endocytic machinery associated with the formation of clathrin-coated pits Current Opinion in Cell Biology 2004, 16:162–168
[7,8], (Figure 1c), as well as to an ever-growing list of signaling molecules [9,10]. This article summarizes advances over the past two years in understanding these novel adaptor functions of the b-arrestins.
Desensitization The paradigmatic function of the b-arrestins is their ability to desensitize 7MSRs by sterically blocking their interaction with heterotrimeric G proteins (Figure 1b). In the case of the b2AR, this mechanism was originally documented in a reconstituted system of purified recombinant proteins [4–6]. However, recently Perry et al. [9] demonstrated that the b-arrestins bind cAMP phosphodiesterases of the PDE4D family. Upon activation of the b2AR, b-arrestin2 recruits PDE4D isoforms into a complex with the activated receptor, where it is positioned to degrade cAMP at an enhanced rate. This slows the rate of PKA activation. Thus, b-arrestin not only slows the rate of b2AR/Gsstimulated cAMP generation, but in a coordinated fashion increases the rate of cAMP degradation in proximity to the receptor–adenylate-cyclase signaling unit.
Receptor trafficking Endocytosis of activated 7MSRs is a fairly general phenomenon. It may be mediated by clathrin-coated pits, caveolae or other uncoated vesicles [7,8]. Once internalized, receptors may be dephosphorylated, resensitized and recycled to the cell surface, be targeted to lysosomes for degradation, or engage in additional intracellular signaling. b-arrestins-1 and -2 play a central role in mediating the clathrin-dependent internalization of 7MSRs by serving as adaptors linking the receptors to elements of the endocytotic machinery. These endocytic elements include clathrin [10], the clathrin adaptor AP2 [11], the small G protein ARF6 [12] and its guanine nucleotide exchange factor, ARNO, and NSF (N-ethylmelaimide sensitive fusion protein) [13] (Figure 1c). Quite recently, the spectrum of receptors that can utilize b-arrestins for clathrin-mediated endocytosis has significantly expanded. b-arrestin2 was shown to be required for internalization of the 7MSR Frizzled-4 after the receptor’s stimulation by the ligand Wnt5A [14]. In contrast to virtually all previously studied examples, where barrestins are recruited directly to a GRK-phosphorylated receptor, in this case b-arrestin binds to PKC-phosphorylated Dvl2, an adaptor protein previously shown to interact with Fz to mediate its canonical signaling through b-catenin (Figure 2a). Perhaps even more surprisingly, one of the receptors for TGF-b, the single-membrane-spanning TGF-b type-III www.sciencedirect.com
b-arrestins: traffic cops of cell signaling Lefkowitz and Whalen 163
Figure 1
(a) Coupling to G protein
(b) Desensitization / inhibition of G-protein coupling
(c) Internalization
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Classical model of (a) 7MSR activation and signaling, (b) GRK-mediated receptor phosphorylation and b-arrestin mediated desensitization, and (c) b-arrestin-mediated clathrin/AP-2 dependent receptor endocytosis.
receptor, is also internalized by a b-arrestin2-dependent process [15]. It is the only one of more than a dozen different types of TGF-b receptors found to do so when tested. Interestingly, the interaction of b-arrestin2 with the cytoplasmic tail of TbRIII also requires its phosphorylation. However, this is catalyzed by the TGF-b type-II receptor, which is itself a S/T kinase, rather than by a GRK. TbRII and TbRIII then internalize together (Figure 2b). Ubiquitination appears to be centrally involved in barrestin-mediated endocytosis of 7MSRs. b-arrestins bind MDM2 [16,17], an E3 ubiquitin ligase best known for its role in regulating the tumor suppressor p53. When a receptor such as b2AR or the vasopressin receptor is stimulated, it binds b-arrestin, triggering MDM2mediated ubiquitination of the b-arrestin [16,18]. This ubiquitination step is required for b-arrestin to perform its adaptor role in clathrin-mediated endocytosis, although the mechanisms responsible for this have not been elucidated. Interestingly, b-arrestins also act as obligatory adaptors to bring E3 ligases to the receptors, which are also ubiquitinated in a stimulus-dependent fashion [16]. At least in the case of the b2AR, however, this ligase appears not to be MDM2. In contrast with the situation originally described for the yeast STE2 receptor [19], www.sciencedirect.com
receptor ubiquitination is not required for the endocytosis of mammalian 7MSRs. Rather, as shown for the b2AR [16], CXCR4 receptor [20], V2 vasopressin receptor [21] and PAF [22], receptor ubiquitination is necessary for post-endosomal sorting to lysosomes. There are interesting and mechanistically significant differences in the patterns of 7MSR trafficking related to how the receptors interact with b-arrestins. As originally described by Oakley et al., two distinct patterns can be observed [23]. So called ‘class A’ receptors, such as the b2AR, bind b-arrestin transiently, traffic with it to clathrin-coated pits and then dissociate. The receptors then internalize without b-arrestin, and generally recycle relatively rapidly. By contrast, ‘class B’ receptors, such as the AT1A angiotensin II receptor or the V2 vasopressin receptor, bind b-arrestin more tightly and internalize together with it. Such receptors recycle more slowly. Class A receptors tend to bind b-arrestin2 preferentially whereas Class B receptors generally have equal affinity for both forms of b-arrestin [23]. These distinct patterns appear to reflect differing rates of deubiquitination of activated receptor-bound b-arrestin, a process which appears to determine the dissociation of the b-arrestin from the receptors [18]. Class B but not Class A receptors provoke sustained b-arrestin ubiquitination. In fact, when Current Opinion in Cell Biology 2004, 16:162–168
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Figure 2
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(b) Wnt5a Fz4 receptor
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b-arrestin-mediated endocytosis of the Frizzled 4 receptor and non-7MSRs. (a) b-arrestin2-mediated endocytosis of the Wnt5a-stimulated Frizzled 4 (Fz4) receptor mediated by protein kinase C (PLC) phosphorylation of the intracellular b-arrestin adaptor Dishevelled 2 (Dvl2). (b) b-arrestin2-mediated internalization of TGF-b1 receptor subtypes RII and RIII, facilitated by RII phosphorylation of T841 on RIII. (c) b-arrestin-mediated internalization of the IGF1 receptor.
a chimeric molecule consisting of b-arrestin2 with ubiquitin fused in frame to the C terminus, which cannot be deubiquitinated, is transfected into cells, it converts the pattern of endocytosis of class A receptors to that of class B receptors [18]. Perhaps because of their ability to bind b-arrestins more tightly, class B receptors can functionally sequester pools of b-arrestin, thus leading to heterologous regulation of the endocytosis of other receptors. Thus, activation of the NK1 neurokinin receptor inhibits endocytosis of the NK3 receptor [24], and activation of the V2 vasopressin receptor inhibits internalization of the b2AR [25].
Figure 3
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Signaling The most rapidly expanding area of research on the barrestins over the past few years relates to their multifaceted roles as signaling adaptors and scaffolds connecting 7MSRs with an ever-growing list of effector pathways [2,26]. This was initially appreciated in relation to Src family non-receptor tyrosine kinases [27]. Recently, most attention has been directed toward the MAP kinases (Figure 3). These enzymes are downstream elements of highly conserved cascades of MAPKKKs and MAPKKs leading to activation of the MAP kinases, such as the ERKs, JNKs and p38 [28]. Classically, activated MAPKs translocate to the nucleus where they phosphorylate and activate transcription factors. However, a growing list of
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New paradigm for 7MSR signaling, including (a) heterotrimeric-Gprotein-dependent and (b) b-arrestin-dependent mechanisms. There also exists the potential for signaling that is dependent on both heterotrimeric G protein and b-arrestin. www.sciencedirect.com
b-arrestins: traffic cops of cell signaling Lefkowitz and Whalen 165
cytosolic substrates for the MAPKs has been identified in recent years. The heterogeneity and diversity of kinases at each level of the MAPK cascades (for example, there are five ERKs, four p38s and three JNKs) suggested that scaffolding molecules might be used to ensure fidelity and efficiency in activating specific MAPK modules. In the 1990s several such molecules were found, initially in yeast [29] and then in mammalian systems [30]. Recently, it has been found that b-arrestin2 also functions as a scaffold for 7MSR activation of several mammalian MAPKs including ERK1/2 [31–33], JNK3 [34] and at least one of the p38s [35], (Figure 3b). Not only does the b-arrestin form complexes with individual members of a particular MAPK cassette, thereby facilitating activation of, for example, JNK3 by ASK or ERK1/2 by Raf, but it retains the activated MAPK in the cytoplasm, thereby presumably directing phosphorylation of specific cytoplasmic substrates [31–34], (Figure 3b). Coordinately, it inhibits phosphorylation of nuclear transcription factors, thereby actually inhibiting ERK-dependent transcription [36,37]. This ability of b-arrestin2 to retain activated scaffolded MAPKs in the cytoplasm is apparently related to the presence of a leucine-rich nuclear export signal in the C terminus of b-arrestin2, but not of b-arrestin1. This motif appears to mediate nuclear export of b-arrestin2 by a leptomycin-B-sensitive process [38,39]. 7MSR-stimulated b-arrestin-mediated scaffolding and activation of MAPKs appears to be intimately linked to endocytosis of the receptor–b-arrestin complex, although the molecular details of this linkage remain obscure. Class B 7MSRs bind b-arrestins tightly and internalize with them, and are thus much more effective activators of such b-arrestin-scaffolded MAPK pathways [36,37]. In fact, simply switching the C termini between the b2AR (class A) and the V2R (class B) receptors by construction of chimeric molecules is sufficient to change their patterns of b-arrestin binding [40], trafficking [18,40] and MAPK activation [37]. A major question concerning the role of b-arrestins in mediating signaling by 7MSRs is whether they operate in series or in parallel with the classical signal mediator, the heterotrimeric G proteins. Recently, it was demonstrated, at least in the case of the Gq-coupled AT1A angiotensin receptor, that b-arrestin2 can mediate signaling to ERK completely independently of G-protein activation (Figure 3b). This was established using a receptor mutant (DRY AAY) as well as a mutated angiotensin peptide (SII) [41]. Neither the receptor, when activated by Ang II, nor the peptide, when used to activate the WT receptor, are able to activate G proteins. Nonetheless, both stimulate ERK activation and recruit b-arrestin2. The ability of the mutant receptor and of the mutant peptide to activate ERK is completely ablated by treatment of cells with siRNA, which reduces expression of b-arrestin2. Interestingly, 50% of WT–Ang stimulation of ERK www.sciencedirect.com
through the WT receptor is eliminated by the application of siRNA to b-arrestin2, whereas the remaining 50% is blocked by a PKC inhibitor [41]. These findings suggest that two types of pathways mediate ERK activation in response to Ang stimulation, one mediated by G proteins, the other by b-arrestin2 (Figure 3). The results, however, in no way exclude the possibility that in other systems G proteins and b-arrestins might operate sequentially to activate effectors. Another example of apparently Gprotein-independent, b-arrestin-dependent activation of ERK has been described by Azzi et al. [42] It was found that, for both the b2AR and the V2 vasopressin receptor, inverse agonists for Gs-activated adenylate cyclase are in fact positive partial agonists for b-arrestin-dependent ERK activation. Findings such as these also reinforce the idea that there may be multiple ‘active’ conformations of a receptor [43]. In these cases, it would appear that distinct conformations of the receptor are able to activate different types of effectors such as G proteins and b-arrestin2. Such results have significant and obvious implications for the development of novel therapeutic agents. Several examples have also recently been published of b-arrestin-dependent PtdIns-3-kinase/AKT activation. Thrombin, which acts through a 7MSR, stimulates AKT through b-arrestin1-dependent and -independent pathways, with distinct time courses and physiological consequences [44]. The IGF1 receptor, which is itself a tyrosine kinase, was also shown to activate the PtdIns-3kinase/AKT system via a b-arrestin1-dependent process, leading to anti-apoptotic effects [45]. This pathway is independent of the receptor’s tyrosine kinase activity. The molecular details of these signaling pathways remain to be determined. Recently, an interesting example of heterologous regulation of b-arrestin-mediated ERK activation has been presented. Olefsky and colleagues demonstrated that insulin stimulation of rat1 fibroblasts leads to phosphorylation (S412) and ubiquitination of b-arrestin1 [46,47]. After short periods of stimulation, the b-arrestin phosphorylation functionally impairs ERK activation by IGF1, LPA and isoproterenol [46], whereas after longer periods of stimulation ubiquitination of b-arrestin leads to its downregulation, which also impairs ERK activation through a variety of receptors [47].
Chemotaxis Several recent studies indicate an as yet poorly defined role or roles for b-arrestin2 in chemotaxis to a variety of chemoattractants. Splenic lymphocytes (T and B cells) from b-arrestin2 knockout mice show strikingly impaired chemotaxis in vitro to SDF, which is mediated by the CXCR4 receptor [48]. This has also been demonstrated for CXCR4 receptors transfected into HEK293 cells, Current Opinion in Cell Biology 2004, 16:162–168
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where it was further shown that b-arrestin2-dependent activation of p38 MAPK is required for the chemotactic response [35]. The potential pathophysiological significance of these observations has been demonstrated in a mouse model of allergic asthma (ovalbumin sensitization) which does not develop in b-arrestin2 knockout mice, apparently because of a failure of T lymphocytes to accumulate in their airways [49]. Recent findings of Ge et al. suggest that the role of barrestins in chemotaxis may be related to their mediation of MAP kinase activation as described above [50]. Activation of protease-activated receptor-2 (PAR-2) in NIH3T3 cells promotes b-arrestin- and ERK1/2dependent reorganization of the actin cytoskeleton, extension of polarized pseudopodia and chemotaxis. Further, PAR2/b-arrestin/ERK scaffolding complexes are enriched in the pseudopodia [50]. Another mechanism by which b-arrestins may participate in cytoskeletal reorganization during chemotaxis is by binding and translocating Ral GDP dissociation stimulator (Ral-GDS, which promotes GDP dissociation from the small G protein Ral), thereby leading to activation of the Ral effector pathway [51].
Conclusions The b-arrestins have recently emerged as important adaptors and scaffolds linking the activated forms of 7MSRs to a rapidly growing list of cellular signaling systems. They also mediate the clathrin-coated pit internalization of many receptors via their interactions with several components of the endocytic machinery, a process regulated by the ubiquitination and deubiquitination of the b-arrestins. The most thoroughly studied b-arrestindependent signaling system is that leading to ERK activation. The mechanisms by which the b-arrestins scaffold such MAP kinase pathways may be intimately linked to their roles in receptor endocytosis. Physiologic consequences of such b-arrestin dependent, G-proteinindependent signaling are likely to differ from those of G-protein-mediated activation of the same effectors. Delineation of the physiological consequences of barrestin-mediated signaling, as well as of the structural basis for this multi-faceted regulation, is likely in the next few years, as is a further significant expansion of the range of systems in which b-arrestins play important roles.
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18. Shenoy SK, Lefkowitz RJ: Trafficking patterns of b-arrestin and G-protein-coupled receptors determined by the kinetics of b-arrestin deubiquitination. J Biol Chem 2003, 278:14498-14506. The pattern of 7MSR internalization (Class A versus Class B) is determined by the ubiquitination status of b-arrestin. 19. Hicke L, Riezman H: Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 1996, 84:277-287. 20. Marchese A, Benovic JL: Agonist-promoted ubiquitination of the G-protein-coupled receptor CXCR4 mediates lysosomal sorting. J Biol Chem 2001, 276:45509-45512. 21. Martin N, Lefkowitz RJ, Shenoy SK: Regulation of V2 vasopressin receptor degradation by agonist promoted ubiquitination. J Biol Chem 2003, in press. 22. Dupre DJ, Chen Z, Le Gouill C, Theriault C, Parent JL, Rola-Pleszczynski M, Stankova J: Trafficking, ubiquitination and down-regulation of the human platelet-activating factor receptor. J Biol Chem 2003, in press. 23. Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS: Differential affinities of visual arrestin, b-arrestin1, and b-arrestin2 for G-protein-coupled receptors delineate two major classes of receptors. J Biol Chem 2000, 275:17201-17210. 24. Schmidlin F, Dery O, Bunnett NW, Grady EF: Heterologous regulation of trafficking and signaling of G-protein-coupled receptors: b-arrestin-dependent interactions between neurokinin receptors. Proc Natl Acad Sci U S A 2002, 99:3324-3329. The concomitant or sequential heterologous activation of 7MSRs can lead to the sequestration of b-arrestin, depending on the affinity of the receptor for b-arrestin. The functional consequences of receptorstimulated sequestration of the b-arrestins includes decreased subsequent b-arrestin-mediated receptor desensitization and internalization, and presumably decreased signaling. 25. Klein U, Muller C, Chu P, Birnbaumer M, von Zastrow M: Heterologous inhibition of G-protein-coupled receptor endocytosis mediated by receptor-specific trafficking of b-arrestins. J Biol Chem 2001, 276:17442-17447. 26. Perry SJ, Lefkowitz RJ: Arresting developments in heptahelical receptor signaling and regulation. Trends Cell Biol 2002, 12:130-138. 27. Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK et al.: b-arrestin-dependent formation of b2-adrenergic-receptor– Src-protein-kinase complexes. Science 1999, 283:655-661. 28. Marinissen MJ, Gutkind JS: G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci 2001, 22:368-376. 29. Printen JA, Sprague GF Jr: Protein–protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics 1994, 138:609-619. 30. Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma M, Davis RJ: The JIP group of mitogen-activated protein kinase scaffold proteins. Mol Cell Biol 1999, 19:7245-7254. 31. Luttrell LM, Roudabush FL, Choy EW, Miller WE, Field ME, Pierce KL, Lefkowitz RJ: Activation and targeting of extracellular-signal-regulated kinases by b-arrestin scaffolds. Proc Natl Acad Sci U S A 2001, 98:2449-2454. 32. DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW: b-arrestin-dependent endocytosis of proteinaseactivated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 2000, 148:1267-1281. 33. DeFea KA, Vaughn ZD, O’Bryan EM, Nishijima D, Dery O, Bunnett NW: The proliferative and antiapoptotic effects of substance P are facilitated by formation of a b-arrestindependent scaffolding complex. Proc Natl Acad Sci U S A 2000, 97:11086-11091. 34. McDonald PH, Chow CW, Miller WE, Laporte SA, Field ME, Lin FT, Davis RJ, Lefkowitz RJ: b-arrestin 2: a receptor-regulated www.sciencedirect.com
MAPK scaffold for the activation of JNK3. Science 2000, 290:1574-1577. 35. Sun Y, Cheng Z, Ma L, Pei G: b-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol Chem 2002, 277:49212-49219. These findings clearly support the role of b-arrestins as signaling molecules involved in the complex process of CXCR4-stimulated chemotaxis. 36. Tohgo A, Pierce KL, Choy EW, Lefkowitz RJ, Luttrell LM: b-arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. J Biol Chem 2002, 277:9429-9436. 37. Tohgo A, Choy EW, Gesty-Palmer D, Pierce KL, Laporte S, Oakley RH, Caron MG, Lefkowitz RJ, Luttrell LM: The stability of the G-protein-coupled-receptor–b-arrestin interaction determines the mechanism and functional consequence of ERK activation. J Biol Chem 2003, 278:6258-6267. 38. Scott MG, Le Rouzic E, Perianin A, Pierotti V, Enslen H, Benichou S, Marullo S, Benmerah A: Differential nucleocytoplasmic shuttling of b-arrestins. Characterization of a leucine-rich nuclear export signal in b-arrestin2. J Biol Chem 2002, 277:37693-37701. Scott et al. describe the nucleocytoplasmic shuttling of b-arrestin2, the cytoplasmic localization of which is due to a c-terminal nuclear export sequence. Mutating the nuclear export sequence leads to the accumulation of b-arrestin2 in the nucleus. These studies further demonstrate the co-localization of JNK3 and b-arrestin2 within the cell. 39. Wang P, Wu Y, Ge X, Ma L, Pei G: Subcellular localization of b-arrestins is determined by their intact N domain and the nuclear export signal at the C terminus. J Biol Chem 2003, 278:11648-11653. 40. Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG: Molecular determinants underlying the formation of stable intracellular G-protein-coupled-receptor–b-arrestin complexes after receptor endocytosis. J Biol Chem 2001, 276:19452-19460. 41. Wei H, Ahn S, Shenoy SK, Karnik SS, Hunyady L, Luttrell LM, Lefkowitz RJ: Independent b-arrestin2 and G-protein-mediated pathways for angiotensin II activation of extracellular signalregulated kinases 1 and 2. Proc Natl Acad Sci U S A 2003, 100:10782-10787. These studies are the first to demonstrate 7MSR-stimulated b-arresin-2mediated Erk activation, completely independent of heterotrimeric Gprotein activation. 42. Azzi M, Charest PG, Angers S, Rousseau G, Kohout T, Bouvier M, Pineyro G: b-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G-proteincoupled receptors. Proc Natl Acad Sci U S A 2003, in press. ICI118551 and propranolol, inverse agonists for b2AR-stimulated adenylate cyclase, stimulated b2AR-mediated activation of ERK1/2 independently of Gs/Gi. These studies demonstrate b-arrestin-dependent, heterotrimeric-G-protein-independent b2AR signaling, which suggests ligand-specific receptor conformations and intracellular signaling pathways. 43. Kenakin T: Ligand-selective receptor conformations revisited: the promise and the problem. Trends Pharmacol Sci 2003, 24:346-354. 44. Goel R, Phillips-Mason PJ, Raben DM, Baldassare JJ: a-thrombin induces rapid and sustained Akt phosphorylation by barrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression. J Biol Chem 2002, 277:18640-18648. 45. Povsic T, Kohout TA, Lefkowitz RJ: b-arrestin1 mediates IGF-1 activation of PI-3-K and anti-apoptosis. J Biol Chem 2003, in press. 46. Dalle S, Imamura T, Rose DW, Worrall DS, Ugi S, Hupfeld CJ, Olefsky JM: Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating b-arrestin1. Mol Cell Biol 2002, 22:6272-6285. 47. Hupfeld CJ, Dalle S, Olefsky JM: b-arrestin 1 down-regulation after insulin treatment is associated with supersensitization of b2 adrenergic receptor Gas signaling in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A 2003, 100:161-166. Current Opinion in Cell Biology 2004, 16:162–168
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Stimulation with insulin down-regulates b-arrestin1 leading to an increase in b2AR-stimulated cAMP generation. These studies reveal a mechanism by which insulin receptor stimulation can regulate 7MSR function/regulation by affecting b-arrestin1-mediated receptor desensitization and internalization. 48. Fong AM, Premont RT, Richardson RM, Yu YR, Lefkowitz RJ, Patel DD: Defective lymphocyte chemotaxis in b-arrestin2- and GRK6-deficient mice. Proc Natl Acad Sci U S A 2002, 99:7478-7483. Differential regulation and signaling involved in the complex processes driving CXCL12 chemotaxis by different GRKs and b-arrestin2. These studies demonstrate a cell-type-specific chemotactic defect and a positive regulatory role for GRK6 and b-arrestin2 in mediating the chemotactic responses of T and B lymphocytes. 49. Walker J, Fong A, Lawson B, Savov J, Patel D, Schwartz D, Lefkowitz R: b-arrestin2 regulates the development of allergic asthma. J Clin Invest 2003, in press. b-arrestin2-deficient mice are resistant to the development of allergic asthma. These studies implicate b-arrestin2 in the mediation of immune components thought to play a key role in the development of allergic
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asthma. A deficit in b-arrestin2-dependent T-cell chemotaxis to the lung may underlie the phenotype. 50. Ge L, Ly Y, Hollenberg M, DeFea K: A b-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis. J Biol Chem 2003, 278:34418-34426. The authors demonstrate increased b-arrestin-associated phosphoERK1/2 in pseudopodia during PAR2-induced chemotaxis, suggesting a physiological role for b-arrestin-mediated scaffolding in chemotaxis. 51. Bhattacharya M, Anborgh PH, Babwah AV, Dale LB, Dobransky T, Benovic JL, Feldman RD, Verdi JM, Rylett RJ, Ferguson SS: b-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization. Nat Cell Biol 2002, 4:547-555. b-arrestins bind inactive Ral-GDS in the cytosol. fMLP-receptor stimulation leads to the dissociation of this complex and translocation of active Ral-GDS and b-arrestin to the plasma membrane. Re-association of this complex leads to Ral-GDS inactivation. These studies demonstrate 7MSR-mediated regulation of the Ras GTPase, Ral, by b-arrestin.
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