PerspectiveCell Biology

Arrestin Times for Developing Antipsychotics and β-Blockers

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Sci. Signal.  14 Apr 2009:
Vol. 2, Issue 66, pp. pe22
DOI: 10.1126/scisignal.266pe22

Abstract

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) are the largest group of structurally related proteins encoded by the human genome. As signal effectors and allosteric regulators, GPCRs dynamically recruit not only specific heterotrimeric G proteins but also the cytosolic scaffold proteins, β-arrestin 1 and 2, which were originally thought only to serve as negative regulators of GPCR signaling. Although about half of currently available therapeutics target GPCR function, usually at the ligand-binding, orthosteric site, evidence suggests that β-arrestins may be therapeutic targets themselves. Indeed, a hitherto undiscovered action of various antipsychotics is to inhibit the ability of the dopamine D2 receptor to engage β-arrestin 2 and activate glycogen synthase kinase 3, which may be a target for developing therapeutics for schizophrenia. Also, certain β-antagonists (blockers) used to treat heart failure, such as carvedilol, have the added effect of promoting activation of extracellular signal-regulated kinase through β-arrestin. It seems likely that the structure of β-arrestins allows them to detect different types and conformational states of GPCRs and to respond in functionally distinct fashions by using separate cohorts of signaling proteins, thus generating additional possibilities for therapeutic intervention.

Research into the signaling “wiring diagrams” that control cellular functioning has provided insights into the molecular pathology of disease and identified biomarkers and therapeutic targets. In this regard, G protein–coupled receptors (GPCRs) are the largest group of structurally related proteins encoded by the human genome, with the nearly 1000 members accounting for some 3 to 4% of the total; around half of currently available therapeutics regulate GPCR functioning and around a third target GPCRs directly. GPCRs have a core structure of seven transmembrane helices that undergo a conformational change upon agonist binding (1). This triggers interaction with the appropriate heterotrimeric G protein, causing it to exchange bound guanosine diphosphate (GDP) for guanosine triphosphate (GTP), with consequent dissociation into βγ and GTP-bound Gα complexes that regulate the appropriate effector systems. The β2-adrenergic receptor (β2AR), which couples to Gs to activate adenylyl cyclase, is a paradigm not only in analyzing activation but also in defining desensitization, which constitutes both a short-term uncoupling of the GPCR to its G protein and a long-term receptor down-regulation (2, 3). Rapid desensitization of the β2AR results from its agonist-stimulated phosphorylation by G protein receptor kinases (GRKs), triggering recruitment of cytosolic β-arrestin and physical interdict of β2AR coupling to Gs. Two arrestins found exclusively in visual neurons block the interaction of rhodopsin with transducin, and two nonvisual arrestins called β-arrestin 1 and 2 bind GPCRs and negate coupling to the relevant G protein (2, 3). However, it has become evident that β-arrestins do much more than this single critical job (Fig. 1). They also act as multifunctional scaffold proteins and can transduce signals of their own accord, regulating processes such as cytoskeletal reorganization, chemotaxis, and epithelial cell permeability (46). Indeed, two studies indicate that β-arrestins may be therapeutic targets themselves (7, 8): Masri et al. have shown that various antipsychotic drugs target β-arrestin 2, whereas Kim et al. have shown that a discrete subpopulation of β-AR antagonists (β-blockers) can target β-arrestin signaling.

Fig. 1

Signaling through β-arrestin. GPCRs can adopt various conformations depending on the ligands they bind, their oligomerization state, and their posttranslational modifications. Signaling is achieved by coupling to appropriate G proteins and by binding β-arrestins. However, particular conformations of the GPCR may preferentially switch signaling between pathways involving the G protein, β-arrestin, both G protein and β-arrestin, or other signaling molecules. β-arrestins can associate with different complexes, the compositions of which are determined by GPCR association and status, posttranslational modification, and the type of partners bound, which creates functionally distinct subpopulations. In so doing, these GPCR-associated β-arrestin complexes can generate a range of signals that are further determined by cell type and include activation of ERK, p38 MAPK, and JNK pathways; activation of GSK3β; transactivation of EGFR; and activation of signaling through LIM domain kinase (LIMK) and cofilin. PI3K, phosphatidylinositol 3-kinase.

β-arrestin 1 and β-arrestin 2 are 47-kD, soluble cytosolic proteins sharing 75% identity and are dynamically recruited to agonist-occupied GPCRs. Structural data are available for the elongated, resting state of β-arrestin, in which the relative orientation of these domains (which are separated by a flexible hinge region) is governed by at least three sets of intermolecular interactions (2, 3, 9). Thus, β-arrestins may undergo functionally profound conformational changes upon binding to GPCRs, other partner proteins, after phosphorylation and ubiquitination and, potentially, with small molecules as well (Fig. 1). It is believed that β-arrestin has two GPCR sensor sites: one that identifies altered receptor conformation, such as when agonist binds, and another that identifies the GRK-phosphorylated form of the GPCR (3). Binding at either site likely generates distinct conformational changes in β-arrestin, with binding at both sites engendering an interaction of higher affinity and yet another conformationally distinct state. Thus, functionally different effects may ensue depending on how β-arrestin is recruited, albeit with a common set of consequences, namely, negation of G protein coupling to GPCRs, receptor internalization, and decreased GPCR signaling (Fig. 1). Differences in “inputs” to β-arrestin signaling can then be directed by the receptor type; although GRK phosphorylation of certain GPCRs (β2 and α2 adrenergic, m2 muscarinic, chemokine C-C motif receptor 5, and neuropeptide Y1 receptors) appears critical for the efficient agonist-stimulated recruitment of β-arrestins, this is not the case for other receptors (lutropin, substance P, orexin1, leukotriene B, and 5-hydroxytryptamine 4 receptors) (3). Indeed, the same form of β-arrestin bound to different receptors can exert functionally distinct effects (10). In addition, the dimerization or the oligomerization status of GPCRs can affect their conformation, and this is likely to alter the nature of signals emanating from associated β-arrestins (3, 11, 12). Furthermore, G proteins and β-arrestins may act as allosteric effectors on binding to GPCRs, affecting whether receptor ligands (also called orthosteric ligands) serve as either agonists or antagonists for these two distinct signaling systems (13). Thus, particular GPCRs may exhibit multiple sets of conformations that regulate signaling through the G protein and β-arrestin pathways. Indeed, certain GPCRs can be promiscuous in their coupling to different G protein–regulated signaling systems, as seen for glucagon (14) and 5-hydroxytryptamine 2C receptors (15), as well as the β2-adrenoceptor upon phosphorylation by cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) (16), consistent with conformationally distinct states of GPCRs coupling to distinct signaling pathways (Fig. 1).

β-Arrestins are multifunctional signaling scaffold proteins (24). Indeed, proteomics analysis implies that over 100 different proteins are potentially sequestered in complexes involving β-arrestin 1/2 (17), although it remains to be determined which of these bind β-arrestin directly versus indirectly. Nevertheless, the interaction surface on β-arrestin cannot accommodate the simultaneous binding of all its potential binding partners, suggesting that β-arrestin subpopulations must exist in defined protein cohorts (18, 19). The composition of these subpopulations will be governed by not only steric considerations influencing multiple partner-protein docking, but also the exposure of specific binding sites dependent on the particular conformational state of the β-arrestin subpopulation.

The notion that β-arrestin could act as a signal transducer first emerged from observations that it scaffolds members of the mitogen-activated protein kinase (MAPK) signaling cascade, namely, Src and Raf (20, 21). It was later extended by the findings that β-arrestin binds the Raf and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) module, which activates ERK1/2 (extracellular signal–regulated kinase 1 and 2) and the ASK1 (apoptosis signal-regulating kinase 1) and MKK4 (mitogen-activated kinase kinase 4) module, which activates JNK3 (c-Jun N-terminal kinase 3). However, GPCR activation of these pathways through β-arrestin is complex. In the case of ERK, it is dependent on Src recruitment (20, 21) and is influenced by receptor type, cell background, and dimerization state of β-arrestin (22). Transactivation of the epidermal growth factor receptor (EGFR) through GPCR-coupled β-arrestin signaling can lead to ERK activation (23). It can also occur when PKA phosphorylation of the β2AR switches signaling from Gs to Gi. In cells where the cAMP phosphodiesterase PDE4D5 is present, the Gs-to-Gi switch is controlled by a β-arrestin subpopulation that specifically recruits this isoform (24, 25) to the β2AR so as to control local cAMP concentrations. This recruited cAMP-degrading system thus controls phosphorylation of β2AR by PKA tethered to the receptor by A-kinase anchoring protein 79/150 (AKAP79/150) (26). A cytosolic subpopulation of β-arrestin 2 sequesters PDE4D5 constitutively, which most likely triggers a conformational change in β-arrestin 2, because movement of the extreme C-terminal region of β-arrestin 2 is required in order to expose the PDE4D5 docking site on the N-terminal domain (19). Such a system provides a putative paradigm for an alteration in β-arrestin conformation occurring upon the binding of a partner protein (19). In addition, the breadth of β-arrestin signaling through its scaffolding action encompasses other systems, including Akt (also known as protein kinase B), nuclear factor κB (NFκB), cofilin, RhoA, and p38 MAPK (6). Thus, the conformational state of β-arrestin is pivotal to its signaling properties because it will undoubtedly define the range of the subpopulations made with specific cohorts of partner proteins (Fig. 1). In turn, the conformational state of the GPCR is key in determining that of β-arrestin, an effect whose potential for therapeutic exploitation appears to now have been uncovered (7, 8).

Schizophrenia is a major, debilitating mental illness. Although antipsychotic drugs can ameliorate some symptoms, better therapeutics are needed (27). In order to achieve this, an increased understanding about the various pathologies that underpin schizophrenia is required. Genetic approaches have identified several associated genes, underscoring the complex, multifactorial nature of this disease, where several different primary aberrations may lead to a final pathology (28, 29). The D2 dopamine receptor (D2R), to which the clinical efficacy of many antipsychotic drugs correlates as antagonists, inhibits adenylyl cyclase and thus decreases cAMP accumulation by coupling to Gi/o (27, 30). This suggests a linkage between cAMP and schizophrenia, which has been strengthened by observations that the scaffold protein Disrupted-in-schizophrenia-1 (DISC1) binds the cAMP-degrading phosphodiesterase 4B (PDE4B), an enzyme whose gene is also linked to schizophrenia (31). Agonist occupancy of D2R causes GRK to phosphorylate the receptor, which then recruits β-arrestin, ablating Gi/o coupling and activating glycogen synthase 3β (GSK3β) signaling (32). This occurs because the GRK-phosphorylated D2R recruits a β-arrestin 2 population sequestered with protein phosphatase 2A (PP2A) and active, phosphorylated Akt. However, after its recruitment to the D2R, β-arrestin undergoes a conformational change that is thought to then allow the tethered PP2A to dephosphorylate and deactivate sequestered Akt, which subsequently leads to dephosphorylation and activation of the Akt substrate, GSK3β. Given the suggested links between Akt and schizophrenia (33), Masri and colleagues (7) investigated whether the β-arrestin 2 “switch” in this system was a target for antipsychotic drugs by evaluating their action on the ability of the D2R agonist, quinpirole, to recruit β-arrestin 2. Strikingly, all blocked β-arrestin 2 recruitment with greater potency than their effect on cAMP accumulation (Fig. 2). It seems most likely that the antipsychotics stabilized a certain conformational state of D2R such that it could no longer engage β-arrestin 2. Although the underlying molecular mechanism remains to be ascertained, it may be that the antipsychotics alter D2R oligomerization or D2R phosphorylation by GRK or bind as allosteric modulators of components of the recruited β-arrestin 2 complex. This finding may provide a molecular basis for behavioral observations linking β-arrestin, Akt, and GSK3β to dopamine functioning (30). Thus, examining D2R agonist–stimulated recruitment of β-arrestin 2 may provide a useful means of evaluating or identifying additional antipsychotic therapeutics.

Fig. 2

β-arrestin signaling through the D2R. (A) GRK phosphorylates the agonist-occupied D2R. This recruits β-arrestin (β-arr), which not only sterically ablates Gi/o coupling but activates GSK3β signaling, because a conformational change occurs in β-arrestin on binding to D2R, which allows sequestered PP2A to dephosphorylate and deactivate sequestered Akt. This prevents Akt from phosphorylating and activating GSK3β. (B) Antipsychotic therapeutics prevent the recruitment of β-arrestin to the D2R through an undetermined mechanism, and, thus, the associated Akt remains phosphorylated and able to inhibit GSK3β.

β1AR blockade is used as a first-line therapy for heart failure; however, different β-antagonists (blockers) vary in their clinical efficacy (34), with carvedilol, a non–subtype-selective β-AR antagonist, being particularly effective, although the underlying mechanisms for this have been unclear (35). Kim et al. postulated that some β-blockers might exert actions on β1ARs other than simply antagonizing the activation of adenylyl cyclase by β1AR (8). They screened a panel of 20 β-blockers in human embryonic kidney (HEK) cells transfected with both β1ARs and EGFRs and found that two β-blockers, alprenolol and carvedilol, acted aberrantly by triggering the transactivation of the EGFR as well as modestly activating ERK and Akt (Fig. 3). The cardioprotective actions of both alprenolol and carvedilol were mediated by β-arrestin subsequent to their binding to β1ARs (8). It may be that alprenolol and carvedilol stabilize a particular conformation of the receptor that facilitates signaling through β-arrestin. It will be of interest to see whether these drugs alter either the dimerization state of the receptors or the kinetics of β-arrestin recruitment or whether they cause the recruitment of a subpopulation of β-arrestin with a cargo specific to achieve EGFR transactivation. In this regard, marked differences in response to carvedilol have been reported regarding regulation of adenylyl cyclase, EGFR transactivation, and ERK signaling, depending on cell type and receptor density. Indeed, in cells expressing β2ARs, carvedilol causes GRK phosphorylation, recruits β-arrestin, and activates ERK (35). These data suggest that carvedilol engenders a distinct conformational state of the β2AR that promotes signaling through β-arrestin and acts as an inverse agonist on adenylyl cyclase activation.

Fig. 3

Activation of β-arrestin (β-arr) signaling by select β-blockers. (A) β-Blockers antagonize β1AR stimulation of cAMP generation by adenylyl cyclases (AC). ATP, adenosine triphosphate. (B) However, alprenolol and carvedilol act aberrantly on these receptors by serving as agonists of β1AR-mediated β-arrestin signaling, triggering EGFR transactivation and the activation of ERK1/2. ATP, adenosine triphosphate.

Although β1ARs predominate in the heart, β2ARs are also present but in a functionally distinct compartment (36), which may reflect their differential association with caveolae (37). Thus, each receptor subtype may “channel” cAMP to different PKA subpopulations anchored at spatially distinct sites, leading to different outputs (38, 39). The spatially and functionally separate gradients of cAMP emanating from these receptors are shaped and directed by the different PDE4 cAMP phosphodiesterases that are sequestered with each receptor subtype (16). Indeed, in the heart, β2AR cAMP signaling is predominantly determined by β-arrestin–recruited PDE4D5, whereas β1AR cAMP signaling is predominantly determined by a population of PDE4D8 that directly associates with this receptor (24, 40). ERK signaling is also highly compartmentalized in cells, suggesting that, if carvedilol does stimulate ERK activation and EGFR transactivation, it may be possible to activate distinct pools of effector systems, one associated with the β1AR and the other through the β2AR, with functionally distinct consequences. By targeting β-arrestin, it may be possible to refine β1 antagonists further so as to prevent β1AR overstimulation while providing cardioprotection through β-arrestin–mediated EGFR transactivation.

The domain structure of β-arrestins can detect different types and conformational states of GPCRs and respond in functionally distinct fashions by adopting distinct conformations. These various states likely engender the formation of β-arrestin subpopulations that differ in the cohort of signaling proteins that they recruit, leading to functionally distinct outcomes. β-arrestin subpopulations may also be defined by phosphorylation status, ubiquitination, and perhaps by the first protein that binds to “virgin,” uncomplexed β-arrestin, if it then defines the cohort of proteins that a particular complex can subsequently recruit. Clearly, we need more structural information on changes that occur in β-arrestins upon binding to GPCRs, not only in the agonist-bound, GRK-phosphorylated state, but also for β-arrestin bound to inverse agonists, for ubiquitinated and phosphorylated β-arrestin, and for β-arrestin bound to different partner proteins. Such studies, together with screening of defined β-arrestin complexes, may pave the way to the identification of additional therapeutics and diagnostics.

Acknowledgments

41.Work in the author’s laboratory was supported by grants from the Medical Research Council (G0600765), the European Union (037189), and Fondation Leducq (06CVD02).

References and Notes

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