Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

The non-visual arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.     

Manipulation of Very Few Receptor Discriminator Residues Greatly Enhances Receptor Specificity of Non-visual Arrestins

Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of "receptor discriminator" residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and β(2)-adrenergic receptors (β(2)AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to β(2)AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated β(2)AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to β(2)AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to β(2)AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signaling-biased arrestins targeting the receptor of choice for research or therapeutic purposes.     

Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.     

Selective regulation of endogenous G protein-coupled receptors by arrestins in HEK293 cells

Arrestins play an important role in regulating desensitization and trafficking of G protein-coupled receptors (GPCRs). However, limited insight into the specificity of arrestin-mediated regulation of GPCRs is currently available. Recently, we used an antisense strategy to reduce arrestin levels in HEK293 cells and characterize the role of arrestins on endogenous G(s)-coupled receptors (Mundell, S. J., Loudon, R. B., and Benovic, J. L. (1999) Biochemistry 38, 8723-8732). Here, we characterized GPCRs coupled to either G(q) (M(1) muscarinic acetylcholine receptor (M(1)AchR) and P2y(1) and P2y(2) purinergic receptors) or G(i) (somatostatin and AT1 angiotensin receptors) in wild type and arrestin antisense HEK293 cells. The agonist-specific desensitization of the M(1)Ach and somatostatin receptors was significantly attenuated in antisense-expressing cells, whereas desensitization of P2y(1) and P2y(2) purinergic and AT1 angiotensin receptors was unaffected by reduced arrestin levels. To further examine arrestin/GPCR specificity, we studied the effects of endogenous GPCR activation on the redistribution of arrestin-2 epitope tagged with the green fluorescent protein (arrestin-2-GFP). These studies revealed a receptor-specific movement of arrestin-2-GFP that mirrored the arrestin-receptor specificity observed in the antisense cells. Thus, agonist-induced activation of endogenous beta(2)-adrenergic, prostaglandin E(2), M(1)Ach, and somatostatin receptors induced arrestin-2-GFP redistribution to early endosomes, whereas P2y(1) and P2y(2) purinergic and AT1 angiotensin receptor activation did not. Thus, endogenous arrestins mediate the regulation of selective G(q)- and G(i)-coupled receptors in HEK293 cells.     

The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation

Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.     

Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes

Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. We report the first crystal structure of arrestin-3, solved at 3.0 Å resolution. Arrestin-3 is an elongated two-domain molecule with overall fold and key inter-domain interactions that hold the free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding a wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, which is consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and arrestin-3 we show that the presence of this loose structure is correlated with reduced arrestin selectivity for activated receptors, consistent with a conformational change in this β-sheet upon receptor binding.     

Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains

Agonist stimulation of the β2-adrenergic receptors (β2ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length β2ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized β2ARs in nonlysosomal compartments. Additionally, a lysine-less β2AR (0K-β2AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT β2AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated β2ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the β2AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited β-arrestin2, and internalized just as the WT β2AR. However, only loop 3 and carboxyl tail β2ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the β2AR. We overexpressed and purified the β2AR from HEK-293 cells with or without prior agonist exposure and subjected trypsin-cleaved β2AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the β2AR. These findings introduce a new concept that two distinct domains in the β2AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors.     

The Two Non-Visual Arrestins Engage ERK2 Differently

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. β-arrestin1 and β-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.     

Small ubiquitin-like modifier modification of arrestin-3 regulates receptor trafficking

Nonvisual arrestins are regulated by direct post-translational modifications, such as phosphorylation, ubiquitination, and nitrosylation. However, whether arrestins are regulated by other post-translational modifications remains unknown. Here we show that nonvisual arrestins are modified by small ubiquitin-like modifier 1 (SUMO-1) upon activation of β(2)-adrenergic receptor (β(2)AR). Lysine residues 295 and 400 in arrestin-3 fall within canonical SUMO consensus sites, and mutagenic analysis reveals that Lys-400 represents the main SUMOylation site. Depletion of the SUMO E2 modifying enzyme Ubc9 blocks arrestin-3 SUMOylation and attenuates β(2)AR internalization, suggesting that arrestin SUMOylation mediates G protein-coupled receptor endocytosis. Consistent with this, expression of a SUMO-deficient arrestin mutant failed to promote β(2)AR internalization as compared with wild-type arrestin-3. Our data reveal an unprecedented role for SUMOylation in mediating GPCR endocytosis and provide novel mechanistic insight into arrestin function and regulation.     

Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death

Apoptosis is programmed cell death triggered by activation of death receptors or cellular stress. Activation of caspases is the hallmark of apoptosis. Arrestins are best known for their role in homologous desensitization of G protein-coupled receptors (GPCRs). Arrestins quench G protein activation by binding to activated phosphorylated GPCRs. Recently, arrestins have been shown to regulate multiple signalling pathways in G protein-independent manner via scaffolding signalling proteins. Here we demonstrate that arrestin-2 isoform is cleaved by caspases during apoptosis induced via death receptor activation or by DNA damage at evolutionarily conserved sites in the C-terminus. Caspase-generated arrestin-2-(1-380) fragment translocates to mitochondria increasing cytochrome C release, which is the key checkpoint in cell death. Cells lacking arrestin-2 are significantly more resistant to apoptosis. The expression of wild-type arrestin-2 or its cleavage product arrestin-2-(1-380), but not of its caspase-resistant mutant, restores cell sensitivity to apoptotic stimuli. Arrestin-2-(1-380) action depends on tBID: at physiological concentrations, arrestin-2-(1-380) directly binds tBID and doubles tBID-induced cytochrome C release from isolated mitochondria. Arrestin-2-(1-380) does not facilitate apoptosis in BID knockout cells, whereas its ability to increase caspase-3 activity and facilitate cytochrome C release is rescued when BID expression is restored. Thus, arrestin-2-(1-380) cooperates with another product of caspase activity, tBID, and their concerted action significantly contributes to cell death.     

Distinct Cellular and Subcellular Distributions of G Protein-Coupled Receptor Kinase and Arrestin Isoforms in the Striatum

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.     

Internalization dissociates β2-adrenergic receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β(2)-adrenergic receptors (β(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β(2)ARs between subcellular compartments. BRET between β(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β(2)ARs and endosome markers increases. Energy transfer between β(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β(2)ARs associate transiently with each other in the plasma membrane, or that β(2)AR dimers or oligomers are actively disrupted during internalization.     

A single mutation in arrestin-2 prevents ERK1/2 activation by reducing c-Raf1 binding

Arrestins regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). GPCR complexes with both nonvisual arrestins channel signaling to G protein-independent pathways, one of which is the activation of extracellular signal regulated kinase 1/2 (ERK1/2). Here we used alanine-scanning mutagenesis of residues on the nonreceptor-binding surface conserved between arrestin-2 and arrestin-3. We show that an Arg307Ala mutation significantly reduced arrestin-2 binding to c-Raf1, whereas the binding of the mutant to active phosphorylated receptor and downstream kinases MEK1 and ERK2 was not affected. In contrast to wild-type arrestin-2, the Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation via β2-adrenergic receptor in arrestin-2/3 double knockout mouse embryonic fibroblasts. Thus, Arg307 plays a specific role in arrestin-2 binding to c-Raf1 and is indispensable in the productive scaffolding of c-Raf1-MEK1-ERK1/2 signaling cascade. Arg307Ala mutation specifically eliminates arrestin-2 signaling through ERK, which makes arrestin-2-Arg307Ala the first signaling-biased arrestin mutant constructed. In the crystal structure the side chain of homologous arrestin-3 residue Lys308 points in a different direction. Alanine substitution of Lys308 does not significantly affect c-Raf1 binding to arrestin-3 and its ability to promote ERK1/2 activation, suggesting that the two nonvisual arrestins perform the same function via distinct molecular mechanisms.     

Characterization of G protein-coupled receptor regulation in antisense mRNA-expressing cells with reduced arrestin levels.

Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins have established a role for these proteins in beta2-adrenergic receptor (beta2AR) internalization, desensitization, and resensitization. To validate and extend such findings, we employed an antisense strategy to target the nonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of G protein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targeting arrestin-2 exhibited a selective reduction (approximately 50%) in arrestin-2 levels, while arrestin-3 antisense constructs resulted in reductions (>/=50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cells demonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent of agonist-induced internalization of exogenously expressed beta2ARs, but had no effect on internalization of either m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing the role of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantly decreased the rate of endogenous beta2AR internalization, desensitization, and resensitization. Further analysis demonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels. The effects on the beta2-adrenergic, A2b adenosine, and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction in arrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility of antisense approaches in the examination of arrestin-mediated GPCR regulation.     

Arrestin-2 differentially regulates PAR4 and ADP receptor signaling in platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.     

An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP₆). IP₆ interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP₆ on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP₆, arrestin-2 forms “infinite” chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP₆; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP₆, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP₆ effect. Because IP₆ is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.     

The third intracellular loop of alpha 2-adrenergic receptors determines subtype specificity of arrestin interaction

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the alpha(2b)- and alpha(2c)-adrenergic receptors (ARs) while having no effect on the alpha(2a)AR, here we used alpha(2)ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the alpha(2a)AR, alpha(2b)AR, or alpha(2c)AR. These studies revealed that arrestin-3 directly binds to the alpha(2b)AR and alpha(2c)AR but not the alpha(2a)AR, whereas arrestin-2 only binds to the alpha(2b)AR. Truncation mutagenesis of the alpha(2b)AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the alpha(2b)AR third intracellular loop. Mutation of these residues in the holo-alpha(2b)AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant alpha(2b)AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the alpha(2b)AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.     

Differential roles of arrestin-2 interaction with clathrin and adaptor protein 2 in G protein-coupled receptor trafficking

The non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, beta(2)-adaptin (the beta-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and beta(2)-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated beta(2)-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in beta(2)-adaptin binding and LIELD (residues 376-380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to beta(2)-adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-DeltaLIELD) or beta(2)-adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the beta(2)-adrenergic receptor, while arrestin/beta(2)-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and beta(2)-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated beta(2)-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking.     

Arrestin-mediated signaling: Is there a controversy?

The activation of the mitogen-activated protein(MAP) kinases extracellular signal-regulated kinase(ERK)1/2 was traditionally used as a readout of signaling of G protein-coupled receptors(GPCRs) via arrestins, as opposed to conventional GPCR signaling via G proteins. Several recent studies using HEK293 cells where all G proteins were genetically ablated or inactivated, or both non-visual arrestins were knocked out, demonstrated that ERK1/2 phosphorylation requires G protein activity, but does not necessarily require the presence of non-visual arrestins. This appears to contradict the prevailing paradigm. Here we discuss these results along with the recent data on gene edited cells and arrestinmediated signaling. We suggest that there is no real controversy. G proteins might be involved in the activation of the upstream-most MAP3Ks, although in vivo most MAP3K activation is independent of heterotrimeric G proteins, being initiated by receptor tyrosine kinases and/or integrins. As far as MAP kinases are concerned, the best-established role of arrestins is scaffolding of the three-tiered cascades(MAP3K-MAP2 K-MAPK). Thus, it seems likely that arrestins, GPCRbound and free, facilitate the propagation of signals in these cascades, whereas signal initiation via MAP3K activation may be independent of arrestins. Different MAP3Ks are activated by various inputs, some of which are mediated by G proteins, particularly in cell culture, where we artificially prevent signaling by receptor tyrosine kinases and integrins, thereby favoring GPCR-induced signaling. Thus, there is no reason to change the paradigm: Arrestins and G proteins play distinct non-overlapping roles in cell signaling.     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.