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. Author manuscript; available in PMC: 2022 Oct 17.
Published in final edited form as: Science. 2022 Jul 7;377(6602):222–228. doi: 10.1126/science.abj4922

Conformational selection guides β-arrestin recruitment at a biased G protein-coupled receptor*

Andrew B Kleist 1,2,†,‡, Shawn Jenjak 1,†, Andrija Sente 3, Lauren J Laskowski 4, Martyna Szpakowska 5, Maggie M Calkins 4, Emilie I Anderson 4, Lisa M McNally 4, Raimond Heukers 6,#, Vladimir Bobkov 6, Francis C Peterson 1, Monica A Thomas 1,2,&, Andy Chevigné 5, Martine J Smit 6, John D McCorvy 4, M Madan Babu 7,8, Brian F Volkman 1,$
PMCID: PMC9574477  NIHMSID: NIHMS1828936  PMID: 35857540

Abstract

G protein-coupled receptors (GPCRs) recruit β-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical Chemokine Receptors (ACKRs) are intrinsically-biased GPCRs that engage β-arrestins but not G proteins, making them a model system for investigating the structural basis of β-arrestin recruitment. Here, we perform NMR experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that β-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand binding pocket and intracellular β-arrestin coupling region. NMR studies ACKR3 mutants defective in β-arrestin recruitment, identify an allosteric “hub” in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides β-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

One Sentence Summary:

Dynamic changes to conformational ensembles at intra- and extracellular sites guide β-arrestin recruitment at a biased GPCR

Once thought of as GPCR “off switches”, β-arrestins are now known to coordinate diverse, G protein independent signaling responses (1). In a phenomenon called biased signaling, some GPCR ligands (biased ligands) preferentially recruit β-arrestins (or G proteins (2). Because they select one pathway, and therefore a specific functional outcome, biased ligands have demonstrated advantages over conventional ligands in pre-clinical (3–5) and clinical (6, 7) testing. More than 30% of FDA approved drugs target GPCRs (8), therefore understanding the mechanistic basis by which GPCRs recruit β-arrestins has important implications for drug development.

Recent GPCR-β-arrestin structures (9–11) and biophysical studies of β-arrestin-biased ligands (12–14) provide mechanistic insights into β-arrestin recruitment. For instance, structural comparisons of GPCRs bound to antagonists and β-arrestin biased ligands suggest that ligand-specific conformational changes govern β-arrestin recruitment (12, 13). Despite this progress, how ligand binding pocket changes are transmitted to the β-arrestin interface remains poorly understood. Other findings have complicated our understanding of β-arrestin recruitment by GPCRs. Identification of multiple, distinct β-arrestin recruiting GPCR conformations suggests there may be multiple “conformational solutions” to β-arrestin recruitment (15), but raises the question of how such different conformations could elicit similar outcomes. Conversely, nearly identical conformations of β-arrestin- and G protein-bound GPCRs (9–11) suggest that conformational changes alone might not account for β-arrestin recruitment. Finally, some studies of β-arrestin recruitment employ ligands that provoke residual signaling at G proteins (16), making it challenging to isolate the specific molecular changes leading to β-arrestin recruitment.

Given these complexities, studies of β-arrestin recruitment could benefit from functionally “decoupled” receptors which exclusively recruit β-arrestin. Atypical Chemokine Receptors (ACKRs) represent one such naturally occurring system. ACKR3 is an intrinsically β-arrestin-biased GPCR that recruits β-arrestin but does not activate G protein (Fig. 1A) (17). Here, we perform NMR studies of ACKR3, and show that β-arrestin recruitment at an intrinsically biased GPCR is guided by “tuning” its conformational equilibrium.

Fig. 1. β-arrestin-biased signaling at ACKR3 and structural characterization by NMR.

Fig. 1.

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(A) CXCL12 activates β-arrestin but not G protein at ACKR3. (B) CXCL12-mediated cAMP inhibition of CXCR4 (positive control) and ACKR3 in Glosensor assay (EC50 = 0.21 nM, CXCR4) (top). CXCL12-mediated β-arrestin-2 recruitment to ACKR3 in Tango assay (EC50 = 3.9 nM, ACKR3) (bottom). N=3 in triplicate in both assays; error bars reflect S.E.M. (C) β-arrestin recruitment to ACKR3 via Nano-BiT assay. VUN701 dose response alone could not be fit (black circles). Purple circles reflect VUN701 dose response with CXCL12 at 3.2 nM. See text and Table S1 for EC50 and Emax. All conditions N=3 in duplicate. Error bars reflect S.E.M. (D) Summary of ligand potency at β-arrestin recruitment. (Structures not to scale; CXCL12 PDB: 2KEC; nanobody from PDB 6KNM used to represent VUN701; LIH383 and CCX777 were modeled in PyMol). LIH383 sequence is FGGFMRRK (20). The chemical structure of CCX777 is shown in (19). (E) 1H-13C HSQC NMR spectra of WT-ACKR3 with various ligands at 310 K. Assigned methionine residues are labeled; (*) denotes inferred assignments from other ligand-bound states (Methods). Negative contour peaks shown in semi-transparency and dashed lines. Peaks marked “a” encompass natural abundance peaks from buffer and detergent components (see also Fig. S2G, S3). All spectra are shown at the same contour except LIH383, which was lowered to represent M2125×39.

RESULTS

Ligand-specific conformational changes at ACKR3

In agreement with prior studies, ACKR3 recruits β-arrestin but does not activate G protein signaling (Fig. 1A,B; Fig S1A; Table S1). To investigate the mechanisms underlying β-arrestin recruitment at ACKR3, we first measured β-arrestin-2 recruitment in response to a panel of ACKR3 agonists, including the endogenous chemokine CXCL12 (EC50= 0.75 nM) (18), the small molecule CCX777 (EC50 = 0.95 nM; Emax (% of CXCL12) = 75 ± 2%) (19), and a recently described peptide LIH383 (EC50 = 4.8 nM; Emax= 83 ± 1%) (Fig. 1C) (20). We also identified a potent, extracellularly-targeting ACKR3 competitive antagonist nanobody termed VUN701 (IC50 = 1.47 uM) using nanobody phage display (Fig. 1C, Fig. S1B–F, Methods). These four ligands display a range of activities for β-arrestin-2 recruitment, allowing us to sample inactive (i.e., VUN701-bound) and active (i.e., CCX777-, LIH383-, and CXCL12-bound) β-arrestin recruiting states of ACKR3 (Fig. 1D).

NMR spectroscopy allows simultaneous characterization of receptor conformation at multiple sites (21). We previously purified 13CH3-ε-methionine-labeled ACKR3 bound CCX777 in MNG/CHS micelles for NMR studies (22), and we use this method for other ACKR3-ligand complexes here (Methods, Fig. 1E, Fig. S2, S3). As with other GPCRs, co-purification with ligands was necessary to produce sufficient quantities of ACKR3 for NMR studies (23). Structural homology modeling of ACKR3 (Methods; Fig. S4) demonstrates 8 native methionine residues (excluding the N-terminal methionine) distributed in ACKR3’s tertiary structure for NMR labeling. NMR labels serve as sensitive probes reporting on regional changes in GPCR conformation and dynamics (24, 25). In this setting, Met2125×39 and Met1383×46 (superscripts indicate GPCRdb nomenclature (26)) are well positioned to report on conformational changes associated with β-arrestin recruitment at ACKR3 because they are located in key regions of the ligand binding pocket (27, 28) and intracellular effector binding interface (29), respectively.

Conformational plasticity in the ligand binding pocket modulates β-arrestin recruitment

Ligand interactions with TM5 play key roles in GPCR activation (27, 30), but how conformational changes in TM5 lead to β-arrestin recruitment is unclear. NMR spectra for the four ligand-bound states show chemical shift perturbations (CSPs) for the Met2125×39 peak, which remains collinear along the 1H-axis but variable along the 13C-axis (Fig. 2A,B). This indicates that ACKR3 exists along a two-state equilibrium, with the peaks at either extreme defining the conformational “endpoints” (31). In the CCX777-bound state, downfield peak positions (~18.2–19 p.p.m.) indicate the 13CH3 is in a trans rotamer (“endpoint” 1), whereas in the CXCL12- and LIH383-bound states, upfield peak positions (~16–16.5 p.p.m.) indicate the 13CH3 is in a gauche rotamer (“endpoint” 2) (Fig. 2B, Fig. S5A,B) (22, 32–34).

Fig. 2. Conformational changes in the ligand binding pocket and intracellular region characterize the β-arrestin recruiting state.

Fig. 2.

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(A) ACKR3 model depicting M2125×39 and M1383×46 probes. (B) Overlay of M2125×39 peaks from ligand-bound ACKR3 complexes at 310K. 16–16.5 and 18.5–19 p.p.m. peaks (13C) correspond to gauche and trans rotameric states, respectively. Ligand-specific β-arrestin activity is depicted at right. Open and closed locks depict conformational sampling and restriction, respectively (bottom). (C) Ligand-residue interactions were compared between antagonist and β-arrestin-biased ligand-bound GPCRs (Methods). Comparison of the mean number of ligand contacts with TM5, ECL2, and TM7 residues. * p-value < 0.05; ** p-value < 0.005 (unpaired t-test). (D) AT1R contacts with the ligand TRV023 (β-arrestin-biased; black outline) in TM5, ECL2, and TM7 shown as blue spheres (PDB 6OS1). The position of NMR probe 5×39 in AT1R is shown. Stabilization of TM5-ECL2-TM7 by biased ligands depicted as a “lock” (right). (E) Met1383×46 peaks in all four ligand-bound states. Upfield peak positions (1H: ~1.3 p.p.m.) of M1383×46 among agonist-bound states supports ring-current shifts due aromatic side chain interactions. Peaks marked “a” as in Fig. 1. (F) 3×46-contacts residues at 2×43 and 7×53 exclusively in active state complexes and 6×37 exclusively in inactive-state complexes (Methods). (G) β-arrestin recruitment of WT ACKR3 versus alanine mutants of 3×46-contacting mutants by NanoBiT (N = 3). See also Table S1.

In contrast, in VUN701-bound ACKR3, the Met2125×39 peak appears between the two “endpoints” at the random coil 13CH3 position, indicating fast conformational exchange between gauche and trans rotamers in the inactive, β-arrestin-non-recruiting state (Fig. 2B). Indeed, CSPs of Met2125×39 from 13CH3 random coil vary by ligand type (Fig. S5A), suggesting that β-arrestin recruitment is associated with decreasing exchange among Met2125×39 rotamers irrespective of which χ3 rotameric state is selected. Antagonists such as VUN701 may fail to constrain the region sampled by Met2125×39, preserving conformational heterogeneity (seen by increased exchange relative to the active state) and precluding TM5 binding pocket stabilization required for ACKR3 activation. Binding data show that alanine mutagenesis of Met2125×39 similarly affects CXCL12 and VUN701, suggesting that NMR changes between inactive and active states likely represent functionally important regional conformational changes, as opposed to differences in direct ligand contacts at the probe residue (Figure S5C–E; Table S1). Indeed, Met212Ala5×39 has equivalent efficacy for β-arrestin recruitment as WT (Fig. S5F), indicating that Met2125×39 reports on but does not itself mediate functional changes in the TM5 region.

Does this occur in other GPCRs? We identified all ligand-contacting residues among structures of (i) GPCRs bound to β-arrestin-biased ligands (or full length β-arrestin; jointly termed β-arrestin recruiting states) and (ii) inactive-state structures of the same GPCRs (Fig. S6A,B). Using contact network analysis (29, 35), we found that ligands in β-arrestin recruiting states make similar numbers of contacts with residues in TM5, but significantly more contacts with residues in ECL2 and TM7 than do antagonists (Fig. 2C). Thus, TM5 may “anchor” agonist interactions with ECL2 and TM7 (27, 36), creating a tripartite “lock” involving the 5×39 region (Fig. 2D; Fig. S6C), whereas antagonists fail to stabilize all three “lock” points (ECL2, TM5, TM7) simultaneously.

Conformational restriction at the β-arrestin coupling region in the active state

Large scale conformational changes underlying GPCR activation are driven by local rearrangements of residues at key positions in the GPCR structure (so-called microswitch residues (37). We next examined Met1383×46, a microswitch residue in the β-arrestin coupling region (Fig. 2A) (29). In the three agonist-bound states (CXCL12-, CCX777-, and LIH383-bound), the Met1383×46 NMR peak has nearly identical chemical shifts (Fig. 2E), indicating that all three agonists elicit a shared conformation of Met1383×46. The upfield position (~1.3 p.p.m.) of Met1383×46 in the 1H-dimension likely reflects ring current shifts caused by proximity to an aromatic side chain (33, 38, 39). These data indicate that the agonist-specific conformations of Met2125×39 (i.e. trans vs. gauche) in the TM5 binding pocket are “funneled” into a common conformation at the β-arrestin coupling region sampled by M1383×46.

In contrast, the inactive, VUN701-bound ACKR3 state is characterized by CSPs of the Met1383×46 peak in both 1H- and 13C-values (Fig. 2E), indicating diminished ring-current shift (1H-dimension), depletion of the gauche rotamer (13C-dimension), and more gauche/trans exchange (13C-dimension). As in the binding pocket, VUN701 causes the smallest CSP from random coil 13CH3-ε-methionine versus the three agonists, suggesting that agonists regulate β-arrestin recruitment by decreasing conformational heterogeneity in this region of the β-arrestin coupling region relative to the inactive state (Fig. S7A).

What is the role of 3×46 – a known microswitch involved in G protein activation (29) – in β-arrestin recruitment among other GPCRs? We calculated all intramolecular, residue-residue interactions among structures of (i) β-arrestin-bound and (ii) inactive-state structures of GPCRs with resolved side chains (β1AR and rhodopsin) (Fig. S7B,C), finding that 3×46 contacts 2×43 and 7×53 in β-arrestin-bound structures and 6×37 in inactive structures (Fig. 2F; Fig. S7D–F). Of note 7×53 is a highly conserved tyrosine among class A GPCRs (37), and 7×53 and 6×37 have been shown to stabilize GPCRs in G protein signaling (7×53) and inactive states (6×37), respectively (29).

To investigate whether 3×46 and active-state contacts from GPCR-β-arrestin complexes (2×43 and 7×53) might play a role in β-arrestin recruitment to ACKR3, we tested Met138Ala3×46, Ile84Ala2×43, and Tyr315Ala7×53 mutants for β-arrestin recruitment (Fig. 2G; Table S1). While Met138Ala3×46 and Ile84Ala2×43 minimally affect ACKR3 function, Tyr315Ala7×53 almost completely abolishes β-arrestin recruitment, demonstrating the essential role for this residue on ACKR3 activation. The close proximity of 3×46 to Tyr7×53 (Fig. 2F; Fig. S7D–F) and the pronounced ring current shift (1H) of Met1383×46 (Fig. 2E) in active- but not inactive states of ACKR3 suggest that Met1383×46 is reporting on dynamic alterations in Tyr3157×53 associated with β-arrestin recruitment (33, 38, 39). Active-state interactions between 3×46 and 7×53 may thus “lock” this region in place, decreasing local conformational heterogeneity and increasing the likelihood of β-arrestin engagement. Indeed, receptors contact the β-arrestin finger loop – a key structural region anchoring GPCR-β-arrestin interactions – directly at or in the vicinity of 3×46 and 7×53 in β1AR- and rhodopsin-arrestin complexes (11, 40). In contrast, a more heterogeneous inactive state (characterized by rotameric exchange and diminished ring current shift at Met1383×46) may reflect absent interactions with 7×53 and a lower propensity for β-arrestin engagement (Fig. 2F).

Allosteric coordination of intracellular and extracellular exchange by the receptor core

To what extent are dynamic changes in extracellular (Met2125×39) and intracellular (Met1383×46) probes allosterically linked through the receptor core during ACKR3 activation? Conserved polar core residues including the residue Asn3×35 (12, 41, 42) coordinate a sodium ion (inactive state) and a water network (active state) to regulate GPCR activation and bias (Fig. 3A) (42). We investigated the effects of known inactivating and constitutively activating mutations of conserved polar core residue Asn1273×35 on ACKR3 activation. Consistent with prior results, the Asn127Lys3×35 mutation showed complete inactivity in β-arrestin recruitment (Fig. 3B), whereas Asn127Ser3×35 acts as a constitutively active mutant (CAM) (Fig. 3B; Table S1) (43).

Fig. 3. Mutational inactivation of β-arrestin recruitment through the conserved polar network.

Fig. 3.

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(A) Position of Asn1273×35 in ACKR3 model relative to Met2125×39 and Met1383×46. (B) β-arrestin-2 recruitment with CXCL12 for ACKR3 mutants Asn127Lys3×35 and Asn127Ser3×35 in Tango assay. All conditions N=3 in triplicate. Error bars reflect S.E.M. See also Table S1. (C) Closeup of 1H-13C-HSQC in Met2125×39 region (right), highlighting VUN701-WT-ACKR3 (first panel), CXCL12-ACKR3 Asn127Lys3×35 (second panel), CXCL12-wild type (WT)-ACKR3 (third panel), and CXCL12-ACKR3 Asn127Ser3×35 (fourth panel). (D) Overlay of 13C-HSQC in the Met1383×46 region for WT-ACKR3-VUN701, ACKR3-Asn127Lys3×35-CXCL12, ACKR3-Asn127Ser3×35-CXCL12, and WT-ACKR3-CXCL12 complexes. A shaded triangle suggests peak non-collinearity. Peaks marked “a” as in Fig. 1. See S8A for Met1383×46 assignment method. (E) Normalized CSPs for Met2125×39 in 13C-dimension (y-axis) and Met1383×46 in 1H-dimension (x-axis) from random coil for Asn1273×35 mutants and CXCL12- and VUN701-bound WT-ACKR3. Arrows depict transitions among CXCL12-bound WT- and mutant ACKR3. (F) Depiction of differences between WT-ACKR3 and Asn127Lys3×35 in CXCL12-bound states. Despite being bound to CXCL12 (blue key), Asn127Lys3×35 “locks” ACKR3 in the inactive state, abrogating CXCL12’s effects on Met2125×39 and Met1383×46 probes. (G) Comparison of residue-residue interactions AT1R-β-arrestin biased ligand and -antagonist bound states (top) reveals formation of a 3×39–7×46 interaction in the β-arrestin state (bottom).

How do Asn1273×35 mutants affect conformation and stability of the binding pocket and intracellular probes as observed by NMR chemical shifts? In the context of CXCL12-bound, the Asn127Lys3×35 mutation shifts the Met2125×39 peak downfield relative to its position in the wild type (WT)-ACKR3 complex (Fig. 3C; Fig. S8A), resembling the VUN701-bound state characterized by trans/gauche rotamer exchange at Met2125×39. This reveals that the receptor core mutation exerts long range, allosteric effects that enhance conformational exchange in the extracellular ligand binding pocket. The Asn127Lys3×35 mutation shifts the Met1383×46 peak downfield in both the 1H- and 13C-values to an intermediate position between those in CXCL12- and VUN701-bound states (Fig. 3D). This peak is not collinear with CXCL12 and VUN701, suggesting occupancy of a third, distinct conformation in this ACKR3 mutant. While Met1383×46 in this mutant occupies a gauche rotamer (13C: ~16.1 p.p.m.), its 1H-value is closer to that of VUN701-bound ACKR3, suggesting fewer local aromatic interactions resulting in a diminished ring current shift. We speculate that this downfield 1H shift reflects increased conformational exchange between both gauche rotamers (i.e. + and −) in the mutant state, which would weaken interactions with an aromatic side chain as compared to a single gauche rotamer (i.e. + or −) in the active state (Fig. S8B). Regardless, pronounced downfield 1H shifts and peak broadening of Met1383×46 in the CXCL12-bound, inactivating Asn127Lys3×35 mutant reveal that, as in the ligand binding pocket, the conserved core mutation abrogates β-arrestin-2 recruitment by increasing conformational heterogeneity at a key position in the β-arrestin coupling region (Fig. 3E). Thus, a single mutation in the receptor core, Asn127Lys3×35, destabilizes ACKR3 at extracellular and intracellular sites, overriding the effects of CXCL12 to decrease conformational exchange present in WT ACKR3 (Fig. 3F).

In the CXCL12-bound, constitutively active Asn127Ser3×35 mutant receptor, Met2125×39 and Met1383×46 peaks overlay with those in the CXCL12-WT-ACKR3 state (Fig. 3C–E, Fig. S8A) despite its unresponsiveness to CXCL12, indicating that the activating mutation promotes conformational homogeneity (no rotameric exchange) extracellularly and intracellularly, even as it decouples allosteric transmembrane communication. In contrast, VUN701 decreases the elevated basal β-arrestin recruitment of ACKR3 Asn127Ser3×35, suggesting that it acts as an inverse agonist at this mutant (Fig. S8C; Table S1). By NMR, the VUN701-bound Asn127Ser3×35 mutant reverts to the inactive (i.e. trans/gauche interconverting) state at Met2125×39, and the Met1383×46 peak is diminished, suggesting transition to a more dynamic state at both probes (Fig. S8D–G).

Among analyzed GPCR structures (i.e. bound to β-arrestin or β-arrestin-biased ligands), AT1R is most closely related to ACKR3 and is the only one that shares Asn3×35. Calculation of intramolecular, residue-residue interactions of AT1R bound to antagonists and β-arrestin biased agonists reveals that while 3×35 interacts with 7×46 (a key residue for β-arrestin recruitment (12) in both states, 7×46 interacts with 3×39 (also involved in β-arrestin recruitment (12) only in the active state (Fig. 3G). This rearrangement in turn, shifts the “register” of TMs 3 and 7 relative to one another in the region above 3×46 and 7×53 (Fig. 3G), suggesting a mechanism by which 3×35 mutations in ACKR3 might be transmitted to the β-arrestin coupling region as observed at the intracellular probe Met1383×46. In effect, Asn3×35 mutations in ACKR3 may function by promoting (active) or disrupting (inactive) 3×39–7×46 interactions, resulting in decreased (active) or increased (inactive) conformational exchange at the intracellular probe.

DISCUSSION

Some studies have identified distinct conformational changes associated with β-arrestin recruitment (12, 13), but others have shown multiple β-arrestin competent conformations (14). How can β-arrestin be both sensitive to and tolerant of GPCR conformational changes?

The results presented here help to reconcile this apparent contradiction by revealing that while β-arrestin is tolerant of diverse GPCR conformations in some parts of the receptor, it may have more stringent conformational requirements in other parts. At TM5 of the ligand binding pocket, we find that multiple “conformational solutions” (15) are compatible with β-arrestin recruitment. These multiple binding pocket conformations are funneled into a single active conformation as monitored at the intracellular probe Met1383×46. NMR and structural evidence for active state interactions between 3×46 and 7×53 suggest that the stringent intracellular conformational requirements comprise the intracellular regions of TMs 3 and 7. Indeed, while studies of AT1R bound to multiple β-arrestin agonists revealed diverse intracellular conformations, most variation was in TMs 5 and 6, with only modest variation in TM7 (14).

Conformational control, while important, may not fully account for β-arrestin recruitment. Observation that intracellular and extracellular probes both transition between conformational heterogeneity (inactive) and homogeneity (active) suggests that β-arrestin recruitment is governed, in part, by tuning the conformational spectrum sampled by the receptor. Regulation of β-arrestin recruitment in this manner could help explain the apparent agnosticism of β-arrestin for specific GPCR conformations (14): alteration of receptor stability in key regions may tune a GPCR’s ability to couple β-arrestin irrespective of conformation (e.g. at Met2125×39). Likewise, globally different GPCR conformations may be similarly tuned to couple β-arrestins by decreasing conformational heterogeneity of the same key epitopes of the β-arrestin interface.

Ligand-specific modulation of the conformational spectrum observed at NMR probes supports a role for conformational selection in β-arrestin recruitment. In this model, a pre-existing spectrum of conformations is narrowed upon perturbation of the system (44), in this case, by addition of various ligands. In the ligand binding pocket, the inactive conformation of M2125×39 is a composite of multiple active conformations, suggesting that β-arrestin recruitment is governed not by switching between distinct “on” and “off” conformations, but by selecting one of many active conformations (active) versus facilitating rapid exchange between them (inactive). The same is only partially true intracellularly. Met1383×46 exists as a composite of trans/gauche rotamers in the inactive state that is narrowed to gauche-only rotamers in the active state. Nevertheless, Met1383×46 only experiences ring current shift effects in the active state. While this might suggest that the active-state conformation of Met1383×46 is not accessible to ACKR3 in the inactive state, it could also reflect sensitivity limitations of 13C as compared to other NMR nuclei (45).

How might different ligands enhance or suppress changes in conformational plasticity to regulate β-arrestin? β-arrestin agonists might be described as stabilizing a particular set of conformations that facilitate β-arrestin binding (Fig. 4). Indeed, contact network analysis of inactive and active state structures of GPCRs reveals enhanced intermolecular (ligand-residue) and intramolecular (residue-residue) interactions in β-arrestin active states, suggesting that β-arrestin agonists might stabilize specific conformations by organizing denser contact networks. Antagonists fail to fasten these locks, leaving them “open” (or conformationally heterogenous), which might disfavor β-arrestin recruitment (Fig. 4).

Fig. 4. Dynamic control of β-arrestin recruitment to ACKR3.

Fig. 4.

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Allosteric regulation of GPCR β-arrestin activation by coordinated transitions in conformational heterogeneity. Ligands constrain (agonists) or promote (antagonists) conformational heterogeneity by altering intermolecular (ligand-residue) and intramolecular (residue-residue) interactions throughout the GPCR structure.

While NMR data and structural analysis point to the association of β-arrestin recruitment with increased stability, other studies show that G protein activation is associated with conformational heterogeneity at the intracellular region (46). There are several possible reasons for this difference. Principally, as compared to extensive GPCR-G protein interface (mediated primarily via the α5-helix of G protein), GPCR-β-arrestin interactions are mediated by a smaller, less structured finger loop. Other reasons include dependence of G protein (but not β-arrestin) engagement on outward, destabilizing motions of TM6 (14, 25), and requirement of G protein (but not β-arrestin) coupling to be linked to GTP hydrolysis.

We propose roles for receptor conformations and conformational equilibria in regulating β-arrestin recruitment, but other aspects of β-arrestin coupling must also be considered. For instance, the observed conformational changes might lead to differences in GRK recruitment between antagonists and agonists, resulting in distinct phosphorylation patterns that alter GPCR functional properties (47). Indeed, β-arrestin interaction with phosphorylated GPCRs in the absence of core engagement has been shown to be sufficient for GPCR internalization and β-arrestin-mediated signaling (but not G protein desensitization) (48). Nevertheless, other studies have correlated the extent of allosteric coupling between agonists and β-arrestin with a particular ligand’s efficacy in β-arrestin recruitment (49), suggesting at least some role for GPCR conformational changes dictating β-arrestin engagement.

In summary, we identify that coordinated, allosterically linked changes in receptor dynamics regulate β-arrestin recruitment to the intrinsically biased receptor ACKR3. Our results provide a framework to understand the molecular changes required for β-arrestin recruitment and provide insights that may facilitate the design of biased therapeutics.

Supplementary Material

1

Acknowledgments:

We thank Acacia Dishman for her comments on the manuscript, and James Campbell and colleagues at Chemocentryx for supplying CCX777.

Funding:

National Institutes of Health grant F30CA196040 (ABK)

National Institutes of Health grant R01AI058072 (BFV)

National Institutes of Health grant F30HL134253 (MAT)

National Institutes of Health grant R35GM133421 (JDM)

State of Wisconsin Tax Check-Off Program for Cancer Research and the Medical College of Wisconsin Cancer Center (BFV)

NIH T32 GM080202 to the Medical Scientist Training Program at MCW (ABK, MAT)

Luxembourg National Research Fund (Pathfinder “LIH383”, INTER/FWO “Nanokine” grant 15/10358798 (AC)

INTER/FNRS grants 20/15084569, PoC “Megakine” 19/14209621, AFR-3004509 and PRIDE 11012546 “NextImmune” (AC)

F.R.S.-FNRS-Télévie grants 7.4593.19, 7.4529.19 and 7.8504.20 (AC)

European Union’s Horizon2020 MSCA Program grant agreement 641833 (ONCORNET) and 860229 (ONCORNET2.0) (MJS)

American Lebanese Syrian Associated Charities (ALSAC; MMB)

UK Medical Research Council (MRC; grant number MC_U105185859; MMB and AS)

Footnotes

Competing interests: BFV and FCP have an ownership interest in Protein Foundry, L.L.C. RH is CSO of QVQ Holding BV. A patent application has been filed on Novel Selective ACKR3 Modulators And Uses Thereof (Applicant: Luxembourg Institute of Health; inventors: AC and MS; PCT Application number: PCT/EP2020/061981).

Supplementary Materials:

Materials and Methods

Figures S1–S7

Table S1

Table S2

References (50–91)

*

This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS.

Data and materials availability:

CCX777 was supplied through a materials transfer agreement (MTA) with Chemocentryx. LIH383 was supplied to BFV through a MTA with AC VUN701 was supplied to BFV through a MTA with MJS All data is available in the main text or the supplementary materials. NMR peak assignments are deposited in the Biological Magnetic Resonance Bank (BMRB) under entries 51451–51454.

References and Notes:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1828936-supplement-1.pdf (1.8MB, pdf)

Data Availability Statement

CCX777 was supplied through a materials transfer agreement (MTA) with Chemocentryx. LIH383 was supplied to BFV through a MTA with AC VUN701 was supplied to BFV through a MTA with MJS All data is available in the main text or the supplementary materials. NMR peak assignments are deposited in the Biological Magnetic Resonance Bank (BMRB) under entries 51451–51454.

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