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ScienceDirect Molecular mechanism of phosphorylation-dependent arrestin activation Martin K Ostermaier1, Gebhard FX Schertler1,2 and Joerg Standfuss1 The past years have seen tremendous progress towards understanding how arrestins recognize phosphorylated G protein-coupled receptors (GPCRs). Two arrestin crystal structures, one of a pre-activated splice variant and one bound to a GPCR phosphopeptide, provided insights into the conformational changes upon phosphate recognition. Scanning mutagenesis and spectroscopic studies complete the picture of arrestin activation and receptor binding. Most perspicuous is the C-tail exchange mechanism, by which the C-tail of arrestin is released from its basal conformation and replaced by the phosphorylated GPCR C-terminus. Three positively charged clusters could act as conserved arrestin phosphosensors. Variations in the pattern of phosphorylation in a GPCR and variations within the C-terminus of different GPCRs may encode specificity to arrestin subtypes and particular physiological responses. Addresses 1 Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen, Switzerland 2 Deparment of Biology, ETH Zurich, Wolfgang-Pauli-Str. 27, 8093 Zu¨rich, Switzerland Corresponding author: Standfuss, Joerg ([email protected])

Current Opinion in Structural Biology 2014, 29:143–151 This review comes from a themed issue on Multi-protein assemblies in signalling Edited by Deborah Fass and Katrin Rittinger For a complete overview see the Issue and the Editorial Available online 5th December 2014 http://dx.doi.org/10.1016/j.sbi.2014.07.006 0959-440X/# 2014 Published by Elsevier Ltd.

Introduction Arrestins form a small family of soluble proteins that regulate a vast number of diverse cell surface receptors, which constitute the G protein-coupled receptor (GPCR) superfamily. GPCRs bind extracellular ligands and undergo conformational changes resulting in G proteindependent intracellular signaling [1]. Once GPCRs are activated, they get phosphorylated by G protein-coupled receptor kinases (GRKs), which facilitate activation and binding of arrestins [2]. Visual arrestins (arrestin-1 and www.sciencedirect.com

arrestin-4) bind rhodopsin and color opsins, while ubiquitously expressed b arrestins (arrestin-2 and arrestin-3) interact at low specificity with the majority of all GPCRs. By binding GPCRs, all four arrestins terminate G protein signaling, while the two b arrestins additionally function as adapter proteins to promote GPCR internalization, degradation, recycling and/or alternative G protein-independent signaling [3,4]. Interaction of arrestin-1 with the dim-light photosensor rhodopsin serves as a prototypical system to study the interaction between an arrestin and its GPCR [5,6] (Figure 1). Already more than 20 years ago, phosphorylation and activation sensors in arrestin-1 have been postulated which would be necessary to fully engage rhodopsin [7]. This theory is based on in vitro GPCR– arrestin binding studies and describes the natural dynamism of GPCRs pragmatically in four states: The phosphorylated or unphosphorylated state both in either inactive or active conformation [7,8]. In this model, arrestin initially exists in a basal conformation which can interact with the phosphorylated C-terminus of its GPCR to form an initial encounter complex [9,10,11,12,13]. This pre-activated arrestin interacts with additional sites in the activated receptor and, potentially, induces further conformational changes to form a high affinity GPCR–arrestin complex. Loss of the agonist does not necessarily imply immediate complex dissociation and a low-affinity complex can be also formed on phosphorylation-independent pathways [11]. Although we still lack direct structural information on GPCR–arrestin complexes, crystal structures of all four arrestins in the basal conformation are available for a while [14–17] and have recently been complemented by crystal structures of the pre-activated splice variant p44 of arrestin-1 [18] and arrestin-2 bound to a receptor phosphopeptide [9] (Table 1). In the following we compare the conformational changes that arrestin undergoes during its activation with recent nuclear magnetic resonance (NMR) [11] and electron paramagnetic resonance (EPR) studies, precisely sitedirected spin labeling (SDSL) [19], site-directed fluorescence labeling (SDFL) [20] and double electron– electron resonance (DEER) [19] studies, as well as with a complete functional map derived from scanning mutagenesis and binding studies of arrestin-1 [21,22]. Current Opinion in Structural Biology 2014, 29:143–151

144 Multi-protein assemblies in signalling

Figure 1

INACTIVE STATES Agonist

INTERMEDIATE Phosphorylationmediated complex

ACTIVE STATES

ACTIVE STATES

INTERMEDIATE

High affinity ligand–receptor–arrestin complex

High affinity receptor–arrestin complex

Phosphorylationmediated complex

Desensitization, siganlling & internalization Current Opinion in Structural Biology

Arrestin activation mechanism. Arrestin undergoes several distinct conformations during its interaction with a GPCR. After mobilization of arrestin from dimeric or tetrameric storage forms [58–60] it is available in its basal conformation (blue) [14–17]. Pre-activated arrestin (violet) is formed by a C-tail exchange mechanism of C-termini from arrestin and activated phosphorylated receptor (R*-P) [9,22]. R*-P engages with its opening cytoplasmic cleft the central loops of arrestin [10,19,20,22,29,30]. This results in complexes of active arrestin (red or green) with ligand-bound or ligand-free activated phosphorylated receptor (R**-P or RApo-P, respectively) [11]. It can be assumed that receptors in high affinity complexes expose an extended cytoplasmic surface [1]. Arrestin likely exists in structurally similar conformations (violet) when associating with or dissociating from different receptor states. Moreover two distinct conformations exist in which monomeric arrestin forms low-affinity encounter complexes with either activated or phosphorylated receptor (R* or R-P, respectively) [11]. Further GPCR–arrestin complexes may co-exist in 2:1 stoichiometry [20,61,62], but were left out for the sake of clarity.

Comparison of pre-activated arrestins All four arrestins share a similar fold characterized by an N-terminal and a C-terminal domain each composed of a seven-stranded curved b-sandwich. The two domains touch each other via loops at the pseudo two-fold rotation axis of the molecule burying a network of charge–charge interactions, called the polar core (Figure 2a,b). The polar core is highly conserved within all four arrestins as exemplified here for arrestin-2. Two asparagine residues, D290 and D297, which are located in the gate loop (residues 290–299), form salt bridges with R169 and R393, which are found in the body of the N-domain or in the C-tail, respectively [15]. Both arginines in turn form hydrogen bonds with D26 located in the N-terminus next to R25, which was suggested to be a central arginine switch [22]

(Figure 2b). The corresponding hydrogen bond network in arrestin-1 is maintained by D296, D303, R175, R382, and D30 [14]. The polar core and the nearby 3-element interaction site, what is named the clamping of the C-tail by a-helix I and b-sheet I (Figure 2b), have been proposed to restrain arrestin in its inactive conformation [23–25]. In this preactivated state, C-domain and N-domain rotate relative to each other by 20–218 [9,18], leading to reorganization of the polar core and release of the arrestin C-tail from the 3-element interaction site. Releasing the arrestin C-tail (383–418 in arrestin-2 and 372–404 in arrestin-1), is crucial for high-affinity binding of arrestin to the activated, phosphorylated GPCR [11,19] and, in case of b

Table 1 List of arrestin crystal structures sorted by PDB release date Arrestin isoform

Conformational state

Arrestin-1 Arrestin-2 Arrestin-1 Arrestin-3 Arrestin-2 Arrestin-2 Arrestin-4 Arrestin-2 Arrestin-2 Arrestin-1 Arrestin-1

Pre-activated Pre-activated Basal state Basal state Basal state Basal state Basal state Basal state Basal state Basal state Basal state

Current Opinion in Structural Biology 2014, 29:143–151

Modification & co-crystallized partner Splice variant p44 Complex with Fab30 & V2Rpp Splice variant p44 Truncation (394-420) Splice variants in complex with clathrin terminal domain Complex with inositol hexakisphosphate – – Truncation (394–418) Glycine inserted at position 2 –

PDBid & publication 4J2Q [18] 4JQI [9] 3UGU [27] 3P2D [16] 3GC3/3GD1 [63] 1ZSH [64] 1SUJ [17] 1JSY [65] 1G4M/1G4R [15] 1CF1 [14] 1AYR [66]

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Phosphorylation-mediated arrestin activation Ostermaier, Schertler and Standfuss 145

Figure 2

Arrestin-2: Inactive conformation

(a)

Middle loop

Finger loop Gate loop

Gate loop

(b)

Small lariat loop

C edge

C tail

F391

Finger loop

R393

Lariat loop

N245 A247

(c)

D135

D297

C tail R169

R25 Polar core 3 Elements

Sweet I Interdomain hinge

Arg switch

R285 K284

D290

D26 N281 C tail

Helix I

R282 Phosphopeptide-bound conformation

(d)

(e)

N domain

(f) N245 Small lariat loop

V2Rpp K11 K10

V2Rpp

K294

R169

K107 C domain 20º

Lariat loop K284 D135

R25

R285 N281

D26 Middle loop

D297 R282

Current Opinion in Structural Biology

Phosphorylation-dependend activation mechanism of arrestin. Arrestin-2 is shown in basal (a–c) and pre-activated state (d–f) (accession code 1G4M (chain A) and 4J2Q, respectively). All residue numbers refer to arrestin-2. The functional map of arrestin-1 [22] is plotted on arrestin-2 for all similar or identical residues. (ClustalW2 aligns bovine arrestin-1 and rat arrestin-2 with 230 fully conserved residues, 82 strongly and 33 weakly similar residues out of a total of 404 or 418 residues, respectively.) Increasing ribbon width and color spectrum ranging from red over white to blue indicate increasing affinity. Functional regions are indicated in the inactive (a) as well as in the V2Rpp phosphopeptide (orange) bound state (d). Arrows in (d) reflect direction and strength of conformational changes with respect to the inactive state (a). R25 in-between the polar core and 3-element interaction site in the basal state (b) interacts with V2Rpp in pre-activated arrestin (e). Residues differing in phosphate binding between arrestin-1 with R*-P and arrestin-2 with V2Rpp are shown in green. Significant loop rearrangements and a 208 twist between N and C domain accompany arrestin activation. The YKS(N)D(A) hydrogen-bond network motif is represented in arrestin-2 by Y63, K138, N245 and A247. It describes interactions between finger, middle and small lariat loop in the basal state (c) and is disrupted during activation of arrestin [18]. New interactions between lariat, gate and middle loop are established. Finger and middle loop residues bind to phosphates in V2Rpp peptide (f).

arrestins, for further downstream events, such as clathrinbinding to the arrestin C-tail for GPCR internalization [26]. Removal of the arrestin C-tail is a prerequisite for highaffinity binding, but is not always sufficient for the transition of arrestin from basal to pre-activated state. For example, the p44 splice variant of arrestin-1 has been crystallized in both basal [27] and pre-activated conformation [18] even though the last 35 residues, including the C-tail, are replaced by a single alanine (Table 1). Apparently the particular crystallization conditions used in either case were able to stabilize both states for structure determination. Nevertheless, in vitro assays show the p44 splice variant clearly to be pre-activated, in the sense that it binds its GPCR rhodopsin independently of phosphorylation [28]. Likely truncation of the C-tail destabilizes the inactive conformation of the polar core by removal of crucial interactions including R382 as www.sciencedirect.com

part of the charged interaction network described above. The p44 and the arrestin-2 pre-activated states show likewise a significant rearrangement of the polar core. The basal state hydrogen-bond network is disrupted and replaced by new interactions except one salt bridge, which is still formed between D26 and R169 in arrestin-2 (D30 and R175 in arrestin-1) (Figure 2e) [9,18]. All other salt bridges within the former polar core are disrupted and allow a significant conformational change of neighboring loops including a rotation of about 1808 observed for the residue at the tip of both the gate loops in arrestin-1 and arrestin-2. Residue K294 in the gate loop (K300 in arrestin-1) can consequently interact with a phosphate moiety of V2Rpp (TP360), a phosphopeptide derived from the C-terminus of the V2 vasopressin receptor (Figure 2e). Residue D297 in the gate loop forms a novel interaction with R282 in the lariat loop (residues Current Opinion in Structural Biology 2014, 29:143–151

146 Multi-protein assemblies in signalling

274–300) and R285 in the lariat loop stabilizes the novel middle loop conformation by a salt bridge with D135 located in the middle loop (Figure 2f) (corresponding interactions in pre-activated p44 are salt bridges between D303 and R288 as well as between R291 and D138). The relative significance of these interactions can be furthermore estimated using the complete functional map of arrestin-1. Overall the reorganization of salt-bridges supports the functional importance of particular loop conformations crucial for receptor interaction. In spite of all similarities between arrestin-1 and arrestin2 pre-activated state structures (SIFig. 1), differences appear in positions where loops interact with phosphate moieties. The finger loop (Figure 2a) adopts an a-helical conformation [19,29] and is dislocated during binding to R*-P [19,30], while its conformational flexibility is reduced upon complex formation [10]. It is anticipated that the finger loop binds within the crevice that opens up on the cytoplasmic receptor face during TM6 and TM5 outwards movement [20,22]. In fact, a tryptophaninduced quenching method showed physical proximity of finger loop mutants Y67W and F79W in arrestin-1 with bimane-labeled position 243 in TM6 of rhodopsin [20] in agreement with data from scanning mutagenesis [22]. Comparative binding of arrestin-1/2 chimeras to R*-P and phosphorylated activated m2 muscarinic receptor further identified the finger loop region as important element determining receptor specificity [31]. Several residues in the finger loop, including R62, Y63 and R65, as well as K138 in the middle loop and residues K77 and R165 in bsheets interact with phosphate moieties (TP347 and SP350) of the phosphopeptide V2Rpp (Figure 2e,f), which do not occur in the rhodopsin C-terminus (Figure 3b). Accordingly, finger loop conformations in pre-activated arrestin-1 and arrestin-2 differ significantly from each other. Although the finger loop in arrestin-1 stretches outwards by extending b-sheets V and VI, the finger loop in arrestin-2 undergoes a similar but less extended motion to allow the interaction of the middle loop with V2Rpp. Mutation of positively charged side chains, which interact with phosphate moieties within the rhodopsin C-terminus, to the methyl moiety of an alanine results in less stable complexes due to hindered interactions. Arrestin-1 mutants R66A (finger loop), K141A (middle loop) as well as R81A and R165A do either not affect interaction with phosphorylated activated receptor (R*-P) or, on the contrary, result in more stable complexes [22]. To some extent, functionalities of corresponding residues in arrestin-1 and arrestin-2 thus depend on the particular interaction partner and its phosphorylation pattern.

Initiation of arrestin activation by a C-tail exchange mechanism Phosphopeptides derived from the C-termini of the V2 vasopressin receptor [26] or rhodopsin [32,33] have been Current Opinion in Structural Biology 2014, 29:143–151

shown to initiate arrestin C-tail release and to allow arrestin binding to the activated, but unphosphorylated GPCR. Also disruption of the 3-element site, in which the arrestin C-tail is clamped, enhances binding to rhodopsin and reduces the selectivity dramatically for non-preferred states of the receptor [23]. Finally, the structure of V2Rpp in complex with arrestin-2 suggests a C-tail exchange mechanism, in which the phosphorylated GPCR C-terminus replaces the arrestin C-tail at overlapping position in the opposite antiparallel b-strand conformation (compare Figure 2a,b with Figure 2d,e) [9]. In addition to the recent structure of b arrestin with bound phosphopeptide, the C-tail exchange mechanism has been supported by a series of potential phosphatesensing residues identified in the functional map of arrestin-1 [22]. These solvent-accessible negatively charged residues include K14 and K15 (in b-strand I), R29, K110 (in helix I) and K300 (in the gate loop (residues 296–305), which is a functional part of the lariat loop (residues 283–305)) [22,23,34,35]. The corresponding residues in arrestin-2 (K10, K11, R25, K107 and K294) indeed undergo charge–charge interactions with phosphates of the V2Rpp peptide (Figure 2e) [9]. The arginine at position 29 interacts either with a conserved phenylalanine in the arrestin C-tail (F380 or F391 in arrestin-1 or arrestin-2, respectively) or with a phosphate in the GPCR C-terminus. The alanine mutant of this arginine bares the weakest binding to the corresponding GPCR so far observed for single point mutations. Located in between the 3-element interaction site and the polar core of arrestin, R29 may control the C-tail exchange mechanism [22]. Nevertheless, not all potential phosphate sensors are conserved throughout the entire arrestin family. This makes physiological sense as the length of GPCR C-termini and the position of phosphorylation sites show considerable variation between different GPCRs. For instance, R7 in arrestin-2 forms contacts to a V2Rpp phosphate [9] and is conserved in arrestin-3 and arrestin-4, but not in arrestin-1 (Figure 2e). On the other hand, R18 and K20 have been shown to act as phosphate sensors in arrestin-1 [17,22,34], but are not conserved in other arrestins. In arrestin-2, R62 at the edge of and R65 in the finger loop (residues 63–75), K138 in the middle loop (residues 129–140) as well as K77 and R165 in b-sheets interact with V2Rpp phosphates (Figure 2f) — however, the rhodopsin C-terminus does not contain phosphorylation sites at the corresponding positions (Figure 3b). Sequence alignments of C-termini from mammalian vasopressins or mammalian rhodopsins including color opsins allow to identify three conserved serine/threonine sites that can interact in their phosphorylated state with three conserved clusters of positively charged amino acids (Figure 3). Namely, these three clusters are formed by www.sciencedirect.com

Phosphorylation-mediated arrestin activation Ostermaier, Schertler and Standfuss 147

Figure 3

(a) 2

bits

1

Vasopressin receptors (b)

(c)

4

bits

2

Opsins Current Opinion in Structural Biology

Arrestin phosphorylation sensors and their interaction partners in GPCR C-tails. The sequences of 15 mammalian vasopressins or 40 mammalian rhodopsins and color opsins were aligned using the Universal Protein Resource alignment tool (www.uniprot.org) and were graphically represented with WebLogo (www.weblogo.berkeley.edu). The regions 382–410 in the vasopressin sequence alignment (a) and 342–370 in the opsin sequence alignment (c) are shown. Residues in arrestin-2 that form charge-charge interactions with phosphates and corresponding phosphorylation sites of the V2 vasopressin receptor C-terminus (V2R) were derived from the structure of arrestin-2-V2Rpp-Fab30 (Protein Data Bank accession code 4JQI). Arrestin-1 phosphate sensors and corresponding phosphorylation sites in the rhodopsin C-terminus (Rho) are described in [22]. The sequence alignment of C-termini from V2 vasopressin receptor and rhodopsin were optimized for overlapping phosphorylation sites (b). The superscript asterisk before arrestin-1 or arrestin-2 residues indicates high conservation in visual or b arrestins, respectively. Additionally, residues at corresponding positions in arrestin-1 and arrestin-2 are displayed in a mirror-like fashion. The arginine in the N-terminus is conserved in positions 7, 8 and 6 in arrestin-2, arrestin-3 and arrestin-4, but not in arrestin-1. Serines and threonines are colored in red and positively charged residues in blue. Light blue colored residues in arrestin-1 or arrestin-2 miss either the corresponding conserved residue ‘partner’ in the other arrestin(-2 or -1) or the interaction partner in the corresponding GPCR C-terminus. Residues are colored in grey in V2R if they are not resolved in the crystal structure. The same color code was used in vasopressin or opsin sequence alignments. In addition, negatively charged or polar residues are displayed in orange or violet and all other residues in black.

residues K14/K110, K15/R29/K300 and K166 in arrestin-1 (and correspondingly, K10/K107, R11/R25/K294 and K11/ K160 in arrestin-2). At this point it is worth noting that, for high affinity binding of arrestin-1, three out of seven phosphorylation sites in the rhodopsin C-terminus need to be phosphorylated [36,37]. It has often been speculated that the position of the phosphates is less important and www.sciencedirect.com

that as long as enough charge is introduced high-affinity binding is promoted [8,36]. The high conservation of the arrestin clusters described above and the relative importance of C-terminal residues to rhodopsin binding resolved in the functional map of arrestin-1 [22] however suggests a model, in which the specific position of phosphates is an important parameter. Current Opinion in Structural Biology 2014, 29:143–151

148 Multi-protein assemblies in signalling

Phosphorylation-dependent activation of arrestin The diversity of arrestin functions together with the observation that ligands can specifically induce G protein-dependent or arrestin-dependent signaling stimulated the development of the so-called phosphorylation barcode theory [38,39,40]. This theory states that biased ligands would stabilize ligand-specific GPCR conformations and thereby induce recruitment of specific GRKs introducing a distinct phosphorylation pattern or ‘barcode’ in the GPCR C-terminus [39]. Subsequent binding of arrestin and adoption of a barcode specific arrestin conformation [41] would determine whether the GPCR is desensitized (including defined arrestin-dependent signaling) or desensitized and internalized [38,40]. Indeed, conserved positions (relative to the NPxxY motif) of serine/threonine clusters in GPCR Ctails have proven to be phosphorylated and essential for formation of stable GPCR–arrestin complexes that can be internalized and stay together within the cell after endocytosis [42]. Arrestin-biased agonists further favor distinct conformational equilibriums in the b2 adrenergic receptor (b2AR) as shown by site-specific 19F-NMR labels at the end of transmembrane (TM) helix 6 and 7 [43]. Principally such distinct conformations could control binding of arrestins either directly or more indirectly by favoring distinct phosphorylation patterns added by different GRKs. GRK1 and GRK2, for example, are able to promote high affinity binding of arrestin-1 to R*-P, whereas GRK5 was much less efficient [44]. Similarly, GRK2 and GRK3 effectively recruited arrestin-3 (also known as b-arrestin 2) for desensitization of vasopressin receptor V2, while GRK5 and GRK6 had much less influence on arrestin-3 recruitment [45]. The V2 vasopressin receptor has a proximal SSS and a distal TSS cluster (shown in red or grey, respectively, Figure 3b). Mutation of the SSS cluster to a triple alanine inhibits receptor internalization, while mutation of the TSS cluster (which seems not to be phosphorylated) does not influence internalization [42]. The SSS cluster is located 36 residues downstream of the NPxxY sequence motif conserved in TM7 of GPCRs. Similarly, neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A receptor and substance P receptor have conserved serine/ threonine cluster motifs 45, 38/47, 25/32 and 50 residues downstream the NPxxY motif [46]. Conversely, GPCRs such as the b2ARs, which are lacking the serine/threonine-rich cluster in conserved proximity to the NPxxY motif, are generally rapidly recycled presumably due to low GPCR–arrestin complex affinity. Conclusively, ligand-induced specific GPCR conformations allow phosphorylation of clusters conserved in their NPxxY-relative position resulting in high affinity GPCR–arrestin complexes that outlast endocytosis. It can be easily imagined from the structure of V2Rpp-bound arrestin-2 and experimentally guided models of the rhodopsin C-terminus arrestin-1 interaction [22], how different phosphorylaCurrent Opinion in Structural Biology 2014, 29:143–151

tion patterns could influence the stability of the corresponding GPCR–arrestin complex even without major structural rearrangements.

Phosphorylation-independent activation of arrestin Although both pre-activated arrestin crystal structures [9,18] show high overall similarity, NMR spectroscopy data argue for the existence of at least four distinct complexes, which would be in the case of rhodopsin: Arrestin-1 in complex with unphosphorylated activated receptor (R*), phosphorylated basal-state receptor (R-P), agonist-free phosphorylated apoprotein (RApo-P), and R*-P [11]. Indeed, fluorescence pump-probe and depolarization experiments have revealed that pre-binding and transition into active state arrestin are temporal separate events [47] (Figure 1). Chemical shifts in NMR of most of the 40% assigned residues [48] can be followed for arrestin-1 upon addition of R* or R-P. Most striking differences between these two complexes emerge for the 3-element interaction site and the C-tail. R* destabilizes the 3-element interaction site (inclusive the proximal C-tail) without displacing the distal C-tail (389–404). By contrast, in presence of low R-P concentration the distal C-tail is displaced and at high R-P concentration the complete C-tail (disruption of the 3-element interaction site included). A large amount of peaks became undetectable due to extensive line broadening upon further titration of R-P, or in presence of R*-P or phosphorylated opsin high-affinity binding to arrestin-1. The authors argue that the loss of signal reflects a molten globule-like structure of the complexes [11]. However, mapping of conformational changes upon R*-P binding with pairs of spin labels in arrestin-1 seem incompatible with a molten-globule model and indicate the main conformational changes to be located in the arrestin loops [19]. In any case it seems likely that several similar arrestin complexes exist, depending on specific activation and phosphorylation patterns.

Conclusions The period 2010–2014 has been filled with breakthroughs in our structural understanding of arrestin-mediated GPCR desensitization. Not only do we have now structures of active GPCRs [49,50,51–55] and pre-activated arrestins [9,18], but the structure of a GPCR–G protein complex [53] further suggests that either interaction partner may undergo significant further conformational changes. One important future question is to what extent the active GPCR states bound to G proteins and arrestins are the same. Mutations in rhodopsin linking TM2 and TM7, for example, increase constitutive activity of the apoprotein opsin [56] towards the G protein but hinder binding of arrestin [44,50] suggesting that both conformations might be quite different. New structures will allow a broader view of the structural variability of a particular GPCR but also about the variability within the whole GPCR family. We also would like to note that www.sciencedirect.com

Phosphorylation-mediated arrestin activation Ostermaier, Schertler and Standfuss 149

during revision of this manuscript two more relevant structural papers appeared. The first describes the structure of opsin with a bound arrestin peptide [67] and the second an arrestin-2-adrenergic receptor complex solved by low-resolution single particle analysis [68]. Both structures are in good agreement with the described phosphorylation-mediated arrestin activation. A better molecular understanding of arrestin-meditated GPCR modulation may promote application of arrestins as natural silencers for pathologically hyper-functioning GPCRs. GPCRs are further the targets for about 30% of small molecular drugs [57] and advanced engineering of arrestins may thus contribute to more robust assays for drug profiling and development.

Conflict of interests statement The authors declare no conflicting interests.

Acknowledgements We are grateful for the financial support from the Swiss National Science Foundation (SNSF) grant 310030_153145 (to G.S.) and 31003A_141235 (to J.S.). We thank Richard Kammerer for critical discussions.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j. sbi.2014.07.006.

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4. Gurevich VV, Gurevich EV: Structural determinants of arrestin  functions. Prog Mol Biol Transl Sci 2013, 118:57-92. The authors give brief insights in a historically grown model of arrestin activation, discuss interactions of arrestins with downstream signaling proteins and introduce arrestin engineering. 5.

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