Pharmacological Reports 66 (2014) 1011–1021

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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep

Review article

Ligand-directed trafficking of receptor stimulus Zdzisław Chilmonczyk a,*, Andrzej J. Bojarski b, Ingebrigt Sylte c a

National Medicines Institute, Warszawa, Poland Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland c Medical Pharmacology and Toxicology, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø – The Arctic University of Norway, Tromsø, Norway b

A R T I C L E I N F O

Article history: Received 25 November 2013 Received in revised form 28 April 2014 Accepted 5 June 2014 Available online 26 June 2014 Keywords: Ligand-directed receptor trafficking Stimulus trafficking Biased agonism Biased signalling

A B S T R A C T

GPCRs are seven transmembrane-spanning receptors that convey specific extracellular stimuli to intracellular signalling. They represent the largest family of cell surface proteins that are therapeutically targeted. According to the traditional two-state model of receptor theory, GPCRs were considered as operating in equilibrium between two functional conformations, an active (R*) and inactive (R) state. Thus, it was assumed that a GPCR can exist either in an ‘‘off’’ or ‘‘on’’ conformation causing either no activation or equal activation of all its signalling pathways. Over the past several years it has become evident that this model is too simple and that GPCR signalling is far more complex. Different studies have presented a multistate model of receptor activation in which ligand-specific receptor conformations are able to differentiate between distinct signalling partners. Recent data show that beside G proteins numerous other proteins, such as b-arrestins and kinases, may interact with GPCRs and activate intracellular signalling pathways. GPCR activation may therefore involve receptor desensitization, coupling to multiple G proteins, Ga or Gbg signalling, and pathway activation that is independent of G proteins. This latter effect leads to agonist ‘‘functional selectivity’’ (also called ligand-directed receptor trafficking, stimulus trafficking, biased agonism, biased signalling), and agonist intervention with functional selectivity may improve the therapy. Many commercially available drugs with beneficial efficacy also show various undesirable side effects. Further studies of biased signalling might facilitate our understanding of the side effects of current drugs and take us to new avenues to efficiently design pathway-specific medications. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ligand-directed trafficking of receptor stimulus (functional selectivity) Ligand and system bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional selectivity in chosen receptor systems . . . . . . . . . . . . . . . . . . b-Adrenergic receptors and heart failure . . . . . . . . . . . . . . . . . . . Calcium sensing receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-HT1A receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dopamine receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m-Opioid receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AC, adenylate cyclase; b-AR, b-adrenergic receptor; CaM, calmoduline; cAMP, cyclic adenosine monophosphate; CHO, Chinese hamster ovary; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; DAG, diacylglycerol; DR, dopamine receptor; GDP, guanosine diphosphate; GIRK, G proteincoupled inward rectifying potassium channel; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; GTP, guanosine triphosphate; h, human; 5-HT, 5hydroxytryptamine; IP3, inositole triphosphate; JNK, Jun N-terminal kinase; MAPK, mitogen activated protein kinase; MEK, MAPK kinase; NF-kB, nuclear factor-kB; PDK, phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PL, phospholipase; PTX, pertussis toxin; TMH, transmembrane helix. * Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Chilmonczyk). http://dx.doi.org/10.1016/j.pharep.2014.06.006 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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Conclusions . . . . . . Funding . . . . . . . . . Conflict of interest . References . . . . . . .

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Introduction GPCRs are seven-transmembrane-spanning receptors conveying specific extracellular stimuli to intracellular signalling. They represent the largest family of cell surface proteins that are directed therapeutically. Nearly 800 different human genes encode receptors for various extracellular ligands including hormones, neurotransmitters and sensory stimuli [1]. More than 30% of the drugs on the market target GPCRs [2]. They contain seven transmembrane helices (TMHs I–VII), three extracellular (E1–E3) and three intracellular (I1–I3) loops, extracellular N- and intracellular C-termini (Fig. 1). It is assumed that upon binding of an agonist, an active receptor conformation is stabilized and the receptor is able to interact with heterotrimeric GTP-binding proteins (G proteins) and cause exchange of GDP to GTP, thereby activating the G protein. According to the traditional two-state model of receptor theory, GPCRs can be considered as operating in equilibrium between two functional conformations, an active (R*) and inactive (R) state. Ligand binding to the receptor alters the equilibrium, with agonists shifting it towards the R* state, inverse agonists shifting it towards the R state and antagonists preventing other ligands (such as endogenous agonists) from binding without altering basal R:R* equilibrium [3,4]. Interaction of an activated receptor with a G protein promotes the release of GDP followed by GTP binding. The G protein-receptor complex is destabilized and the Ga-GTP complex dissociates from the bg heterodimer or, alternatively, a molecular rearrangement occurs [5,6]. Ga-GTP and Gbg subunits are then able to interact with different effector systems. Agonist activation increases the rate of guanine nucleotide exchange and therefore the amount of active Ga-GTP and Gbg. In the inactive conformation, receptors are thought to be functionally uncoupled from the G protein, while the conformational change leading to receptor activation is considered to induce coupling and activation of G proteins. Isomerization between active and inactive conformation

Fig. 1. The architecture of G protein-coupled receptors.

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can also occur spontaneously in the absence of an agonist [7–9]. Signal termination is ensured by the hydrolysis of GTP due to Ga GTPase activity, thereby returning the Ga protein to its basal GDPbound state. In addition to the intrinsic GTPase activity of Ga subunits, the recently discovered regulator of G protein signalling (RGS) family of proteins contribute to Ga-GTP hydrolysis by stabilizing the most favourable conformation of the Ga subunits for GTPase activity [10–12]. Ligand-directed trafficking of receptor stimulus (functional selectivity) Activation of many GPCRs in response to agonists drives signalling in a preferred or canonical pathway (employing mainly adenylate cyclase and phospholipase C) [13]. For instance G protein-dependent activation of serotonin 5-HT1A receptor may involve stimulation of AC, PI3K/Akt, Ras (which may act as a switch between PI3K/Akt and the ERK1/2 pathway) [14,15], PLCb dependent (PI3K independent) caspase 3 inhibition [16] and GIRK [17] activation (Fig. 2). It is now, however, increasingly accepted that receptor stimulation often results in a multitude of signalling outputs and that 7-TM receptors in addition to G proteins can interact with a wide variety of intracellular molecules (such as b-arrestins and kinases) and activate different intracellular signalling pathways [7–9,18–21] (Fig. 3). This may also include receptor desensitization, coupling to multiple G proteins, Ga or Gbg signalling, and pathway activation that is independent of G proteins such as activation of MAPKs family, Src, NF-kB, PI3K. In the classical model, heterotrimeric G proteins mediate signalling via the receptor, while b-arrestins mediate receptor desensitization and internalization. However, it has now been appreciated that b-arrestins can act not only as regulators of GPCRs desensitization, but also as multifunctional adaptor proteins that have the ability to signal through multiple mediators [20,22–24]. Various ligands that affect GPCR indifferent ways and activate specific signalling pathways have been discovered [25,26]. Activation of specific signalling pathways gives agonist ‘‘functional selectivity’’ also called ligand-directed receptor trafficking, collateral efficacy, stimulus trafficking, biased agonism or biased signalling [20,27,28], and involves different subtypes of G proteins or arrestins. Drugs can no longer be classified simply as agonists, partial agonists or antagonists, as it is now recognized that antagonists that block agonist-stimulated receptor activation may also act as inverse agonists to suppress basal receptor activation, or as protean agonists that block one effector pathway but stimulate another (one or more) alternative pathways [23]. A ligand might act as an agonist for one signalling pathway while behaving as an antagonist, partial agonist, or have no effect at all for another signalling pathway of the same receptor. A challenge for new drug development is therefore to discover compounds with high receptor specificity that also can distinguish between the signalling pathways of the particular receptor. The current explanation for biased agonism is that GPCRs can adopt several active conformations stabilized by different ligands. According to the classical receptor model, GPCRs exist in an ‘‘off’’ or ‘‘on’’ conformation causing either no activation or equal activation of all signalling pathways, respectively. Over the past several years it has become evident that this model is too simple [25,26].

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Fig. 2. G protein-dependent signal transduction pathways of the serotonin 5-HT1A receptor.

Different active conformations of a given GPCR affect its signalling pathways distinctly and therefore the signalling profile might vary between various ligands [18,19,21]. It has been proposed that ‘ligands induce unique, ligand-specific receptor conformations that frequently can result in differential activation of signal transduction pathways associated with that particular receptor [29]. Diverse classes of agonists stabilize different receptor conformations that might differ in their ability to interact with G proteins and other receptor-associated proteins such as arrestins which can bind to phosphorylated receptors and function as signalling scaffolds for kinases [2,20,30,31]. In other words, ligand binding to a transmembrane orthosteric site can allosterically alter the receptor conformation that then determines the specificity of binding for the intracellular signalling protein (e.g., G protein or barrestin). This will occur in a ligand-dependent manner, and whether a ligand is considered as an agonist, inverse agonist or

neutral antagonist (rare) depends on the signalling pathways being examined [32]. Taken together, results from different studies support a multistate model of receptor activation in which ligandspecific receptor conformations can differentiate between distinct signalling partners. Inverse agonists of various structures and from different chemical families have been shown to activate multiple signalling pathways such as different protein kinase pathways, gene transcription activation, trafficking, and regulation of cell surface receptor expression [29,33]. It is postulated that GPCR activation dynamics are characterized by a complex equilibrium among multiple conformational states that is manifested in significant basal activity, the existence of allosteric modulators, and a whole spectrum of functional responses including biased signalling. A full understanding of these phenomena requires an ability to probe the equilibrium between different conformations, which is beyond the capabilities

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Fig. 3. G protein-dependent (A) and G protein-independent (B) GPCRs trafficking.

of crystallographic methods that give a highly detailed but ‘‘frozen picture’’ of the lowest energy receptor state [34]. As technology advances to the point at which various behaviours of 7TM receptors can be observed individually, it is clear that rather than being ‘on–off’ switches, 7TM receptors are more akin to ‘microprocessors’ of information. Particular ligands can initiate only portions of the signalling mechanisms mediated by a given receptor. It is assumed that 7TM receptors may be considered as intrinsically disordered and able to adopt many isoenergetic conformations. This idea is consistent with molecular dynamics data which suggest that regions of intrinsic protein disorder are particularly high in cellular signalling proteins. This led to the proposal of a theoretical model in which 7TM receptors exist as clusters of interchanging conformations referred to as ensembles. Ligands produce changes in these ensembles by selective binding to preferred conformations. Therefore, these preferred conformations are stabilized at the expense of other conformations, and a change in the overall collection of 7TM receptor states is produced according to Le Chatelier’s principle (if a dynamic equilibrium is disturbed by changing the conditions, the position of the equilibrium moves to counteract the change) [reviewed in 18].1 Ligand and system bias Biochemical data suggest that the signalling mediated by barrestins has distinct functional and physiological consequences from that mediated by G proteins [36]. The sequence of events connected with G protein-independent b-arrestin mediated ERK activation does not show its typical nuclear localization and is not accompanied by increased activity of the ELK1 transcription factor, what is typically associated with increased phosphorylated ERK 1 This view of pharmacological agonism was described by Burgen [35] as conformational selection.

generation mediated by G proteins [20,37], which then leads to the transcription of immediate-early response genes. At the biochemical level b-arrestins mediated ERK activation has different consequences than G-protein activation. b-Arrestin-mediated phosphorylated ERK is retained in endocytic vesicles [38] in a pathway that is spatially and temporally distinct from activation of phosphorylated ERK by G proteins. In HeK293 cells transiently transfected with the angiotensin 1A receptor, G protein activation of phosphorylated ERK is maximal at early time points (after 2 min), whereas b-arrestin-mediated activity peaks later and is more protracted, accounting for 100% of phosphorylated ERK activity at 30 min [39]. For some receptors, one of the two signalling pathways can translate into beneficial physiological effects, whereas the other appears to mediate the undesirable outcomes. Therefore, depending on the receptor system, it might be possible to develop biased agonists for one of these two pathways into novel and therapeutically more beneficial drugs [40]. For example, the vitamin niacin, a GPR109A agonist which in high doses is used to treat dyslipidemia, appears to exert its triglyceride lowering effect via a G protein-dependent pathway, whereas its side effect of cutaneous flushing depends on b-arrestin 1 [41]. In fact, an agonist of GPR109A which selectively triggers G protein signalling without engaging b-arrestin 1 has been recently described [42]. Concentration–response curves are an accepted method for determining compound activity conveying potency (as a position along the concentration axis) and maximal activity (related to efficacy as the maximal response). From the curves the relative activity of agonists on different signalling pathways can be determined and compared. For a possible bias to be identified it is necessary to separately determine a full concentration–response curve (not a single-point measures of response) for each signalling pathway so that the pathways can be differentiated. System bias reflects the relative efficiency with which different pathways may be coupled to signalling proteins in the cell.

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Signalling G protein

ligandi bias ¼

Biask ¼



b-Arrestin

G1 . . . b 1 Gm . . . b m



Gk

bk

(1)

(2)

Functional selectivity in chosen receptor systems

b-Adrenergic receptors and heart failure Many of these concepts have been explored in detail utilizing adrenoceptors, a therapeutically important group of GPCRs that respond to catecholamines, adrenaline and noradrenaline. b-ARs classically mediate their response to the endogenous ligands (adrenaline and noradrenaline) by coupling to Gas and stimulating cAMP production. However, drugs acting as b-AR agonists or antagonists can activate alternative cell signalling pathways which has been the subject of recent reviews [27,29,46]. After cAMP, the most studied signalling pathways for b-AR ligands are b-arrestin recruitment [47] and ERK1/2 signalling [44,48] but there is also evidence for tyrosine kinase receptor transactivation and p38

G-protein activity

Differences in these efficiencies are related to the relative sensitivity of cellular pathways. For example, in rat atria the cAMP concentration needed to induce myocardial relaxation are lower than the concentrations needed to induce positive inotropy. System bias will be common for all agonists acting on any specific receptor [28]. If a ligand efficacy is detected in a single component functional assay such as a second messenger system, the full texture of that response often cannot be quantified without observing the response in living cells [18]. Therefore, complex detection systems able to detect activities in addition to the so-called ‘primary’ activity of a molecule (for example, b-blockade would be considered as the primary activity of carvedilol) when the molecule is inserted into complex interconnected pathways (that is, whole cells) should be employed [43]. As an example, cellular activities of two biochemically equivalent MAPK14 inhibitors PD169316 and SB203580 may be evoked. In human umbilical vein endothelial cells, these two compounds have opposite orders of potency and efficacy for production of the cell adhesion molecule (P-selectin) versus the production of vascular endothelial growth factor receptor 2, although the biochemical potency of PD169316 and SB203580 is similar (negative log of half maximal inhibitory concentration pIC50 of PD169316 is 7.05 versus 7.30 of SB203580) [44]. Quantifying ligand bias – that is, how much a certain ligand produces bias in a given cellular system may be measured by a plot of b-arrestin against G protein activity [20,45] (Fig. 4). One way to compare signalling between G protein- and b-arrestin mediated pathways is to plot b-arrestin activity on the x-axis and G protein activity on the y-axis. Unbiased ligands would be expected to have equal levels of efficacy for b-arrestin- and G protein-mediated pathways, as shown by the green circles and the line. For biased ligands, there would be differing levels of b-arrestin- and G protein-mediated efficacies, as illustrated for b-arrestin-biased full agonists (grey), b-arrestin-biased partial agonists (yellow), G protein-biased partial agonists (blue) and G protein biased full agonists (magenta). Such data can be also represented in matrix form for m ligands (1) or in terms of a bias factor for each ligand (2) [20].

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β-arrestin activity Fig. 4. Quantifying the pluridimensional efficacies of 7TM receptors. Green circles: unbiased ligands, grey circles: b-arrestin-biased full agonists, yellow circles: barrestin-biased partial agonists, blue circles: G protein-biased partial agonists, magenta circles: G protein biased full agonists (reprinted with permission from Ref. [20]). (For interpretation of the references to color in text, the reader is referred to the web version of this article.)

MAPK, PI3K and NO activation, depending on the b-AR subtype, expression level and cell type [27,32]. Evans et al. [27] reviewed the evidence for ligand-directed signalling for several b-AR pathways including c-Src dependent and c-Src independent ERK1/2 activation, as well as p38 MAPK, PI3K and NOS activation. The recruitment of arrestins, that initiate b2-AR internalization and recycling or degradation, also drives the activation of MAPK pathways. Arrestins function as scaffolds or adaptor proteins for the activation of many signalling networks, including phosphorylation of ERK1/2, c-JNK or p38 MAPK, and other kinases including PI3K and Akt. In mouse embryonic fibroblast cells, b2-AR activation increases ERK1/2 phosphorylation in a biphasic manner. At low agonist concentrations, the response is due to Gas activation, whereas at higher concentrations, c-Src is involved independently of both G proteins and arrestins. In HEK293 cells expressing the b2-AR, c-Src recruitment and subsequent ERK1/2 activation are secondary to binding of arrestin to the receptor. In mouse cardiac myocytes and B lymphocytes b2-ARs have been shown to activate p38 MAPK by a cAMP-PKA-dependent mechanism. However, many other mechanisms may also be involved. b2-ARs activate p38 MAPK utilizing Gbg in HEK293 cells, and b1- and/or b2-ARs activate p38 MAPK through a mechanism involving Gi in rat cardiac myocytes. Activation of p38 MAPK in HEK293 cells by the selective b-adrenoceptors agonist isoprenaline showed biphasic manner: initial (within minutes) activation of p38 MAPK by an arrestin-2/Rac1/NADPH oxidase pathway and delayed activation (at least 90 min) through the cAMP–PKA mediated mechanism [27]. Several b-adrenoceptor ligands have complex efficacy profiles for cAMP generation and ERK1/2 activation at b1-, b2- and b3-ARs [32]. For mouse [49] and human [50] b3-ARs, SR59230A and L748337 are classical competitive antagonists for cAMP accumulation but agonists for ERK1/2 and p38 MAPK activation. Many bblockers are actually weak partial agonists, and others are inverse agonists of the b2-adrenoceptor in a cAMP canonical pathway. At the same time they express their own spectrum of pharmacological properties to activate MAPK or other signalling pathways [32]. For instance, propranolol is an inverse agonist of the b2-AR on the

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Fig. 5. G protein-dependent and b-arrestin-dependent activation of b-AR by propranolol [32].

canonical cAMP pathway, but 41 years after its discovery it was also found to produce a stimulatory ERK1/2 response (Fig. 5) [32,48]. Despite being inverse agonists of AC activity, ICI-118551 also promotes phosphorylation of ERK1/2, and thereby demonstrates its ability to act as agonist of the MAPK cascade, independently of Gs or Gi [48]. Carvedilol, a clinically relevant b2-AR inverse agonist of Gas activation, was found to stimulate Gas-independently of barrestin 2-dependent activation of ERK [47]. ERK1/2 activation by these dual efficacy ligands was not affected by ADP-ribosylation of Gai and could be observed in S49-cyc cells lacking Gas. This indicates that, unlike the conventional agonist isoproterenol, these drugs induce ERK1/2 activation in a Gs/i-independent manner. In contrast, the activation was inhibited by a dominant negative mutant of b-arrestin and was abolished in mouse embryonic fibroblasts lacking b-arrestin 1 and 2. The role of b-arrestin was further confirmed by showing that transfection of b-arrestin 2 in these knockout cells restored ICI118551 promoted ERK1/2 activation. ICI118551 and propranolol also promoted b-arrestin recruitment to the receptor. Taken together, these observations suggest that b-arrestin recruitment is not an exclusive property of agonists, and that ligands classically classified as inverse agonists rely exclusively on b-arrestin for their positive signalling activity [48]. In a study of b1-AR mediated EGFR transactivation, alprenolol and carvedilol were found to induce EGFR internalization. In the heart this process was previously shown to be dependent upon barrestins [51]. Both drugs stimulated EGFR-dependent ERK activation, and were dependent upon phosphorylation of the b1-AR and the presence of both b-arrestin 1 and b-arrestin 2 [18,52]. In a b2-adrenoceptor mutant with poor G protein coupling carvedilol remained a partial agonist for ERK1/2 activation, whereas propranolol produced no response. The carvedilol ERK1/2 response was sensitive to siRNA depletion of arrestin 3, but insensitive to PTX. Carvedilol (but not propranolol) caused receptor phosphorylation, recruitment of arrestin-3-GFP, and receptor internalization without changes in cAMP [32,47]. A study using propranolol (an inverse agonist) and b2-AR-transfected Chinese hamster ovary cells showed that, as expected, basal cAMP production is reduced by propranolol stimulation. However, the

levels of cAMP response element mediated gene transcription were shown to be increased and the increase was dependent on the activation of the p42/p44–MAPK pathway but independent of G protein activation. These results indicate that propranolol could act simultaneously as an inverse agonist, through a Gs-coupled mechanism (decreasing cAMP production), while acting as an agonist by stimulating the MAP kinase pathway through a Gprotein-independent mechanism [43]. It should be noted that b-arrestin-mediated signalling in heart may have important clinical relevance, since it is suggested that the b-arrestin-mediated pathway downstream of the b1-adrenergic receptor protects against cardiomyopathy induced by catecholamine infusion [18,52]. Clinical trials with 16 b-blockers in congestive heart failure indicated that only 3 of them were of benefit, with carvedilol emerging as the best. The data support the idea that it is the additional properties of carvedilol that add therapeutic value to the response to this drug (that is, b2- and a1AR blockade, and antioxidant, antiproliferative and antiendothelin effects) [18,53]. Interestingly, carvedilol is also on the list of bblockers that have b-arrestin mediated ERK-stimulating properties [47]. However, some doubts are raised if the therapeutic benefit of carvedilol in heart failure patients relates to its ability to activate ERK1/2 signalling by G-protein-independent mechanisms [32,47]. It has been suggested that different compounds selectively activate discrete signalling pathways by interacting with particular receptor conformations [32], pointing to a multistate model of receptor activation in which ligand-specific conformations are capable of differential activation of distinct signalling partners [48]. b2-AR conformational studies indicate that there were both quantitative and qualitative differences in the capacity of agonists and partial agonists to induce particular receptor conformations. Differences in Ga coupling was verified by subtype-specific immunoprecipitation of activated Ga subunits labelled with [g-32P]GTP-azidoanilide. For example, the (S,R)-isomer of fenoterol (b2-selective partial agonists with two chiral centres) stimulated substantially higher the activation of Gai2 than did the (R,R)-isomer, while the (R,R)-isomer produced a threefold higher activation of Gas than the (S,R)-isomer. These results are compatible only with the conclusion that the two stereoisomers stabilize distinct conformations of the b2-AR (reviewed in [27]). It was suggested that agonists that selectively stimulate b2-AR Gs coupling, without stimulating b2-AR Gi-coupling or b1-AR activation, may have considerable therapeutic benefit [54]. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) has been used to directly measure changes in the association or relative conformation of bARs and interacting proteins. The data from such studies can then be compared with associated signalling outputs, namely changes in cAMP or ERK1/2 phosphorylation (e.g., Drake et al. [45]). One such study has demonstrated that, relative to isoproterenol, drugs acting as partial agonists for cAMP production can nonetheless act as full agonists for arrestin-3 recruitment. In fact, cyclopentabutanephrine and isoproterenol demonstrate the reversal of efficacy that verifies the presence of ligand-directed signalling, while cyclopentylbutanephrine, a-ethylnoradrenaline and isoetharine are partial agonists for cAMP, they are full agonists for arrestin-3 recruitment [45]. To identify ligand-specific b2-AR conformations, Kahsai et al. [55] examined nine structurally and functionally distinct ligands; propranolol, ICI-118551, carvedilol, isoproterenol, salbutamol, salmeterol, pindolol, carazolol, THRX-144877, each of which had different abilities to signal via a Gas-coupled cAMP pathway, recruit b-arrestins and activate ERK1/2 pathway. Based on their ability to produce receptor-mediated second messenger cAMP, these ligands were classified as full agonists (isoproterenol and

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THRX-144877), partial agonists (salbutamol and salmeterol), weak partial agonist (pindolol), antagonists or inverse agonists (propranolol, carazolol, ICI-118551 and carvedilol). Among the antagonists, both carvedilol and carazolol were able to activate ERK1/2 signalling via G protein-independent mechanisms, whereas carvedilol, but not carazolol, was able to weakly promote barrestin recruitment. Structurally, the four ligands could be grouped as catechol (isoproterenol) or non-catechol (salbutamol, salmeterol, and THRX-144877) arylethanolamine-based agonists, and five (propranolol, pindolol, carazolol, ICI-118551, and carvedilol) were classified as aryloxypropanolamine-based antagonists, commonly referred to as ‘b-blockers’. The data obtained for the b2-AR through a quantitative MSbased strategy suggested that the receptor can exist in multiple conformations that are induced by functionally different ligands. Structurally and functionally distinct ligands exhibited complex patterns of labelling at different residues in the b2-AR, thus providing direct evidence for the presence of multiple ligandspecific conformations. This study therefore provided a definitive and systematic demonstration that different ligands induce qualitatively different receptor conformations. This is consistent with the ability of several structural elements within the receptor to adopt distinct conformations in response to ligands of different chemical structure, which may underlie the recently studied phenomenon of biased agonism and functional selectivity. More broadly, the findings presented here are incompatible with the widely held notion that all ligands of similar functional capabilities stabilize or destabilize similar sets of interactions in a given receptor and with the general concept of ‘two state’ models for receptor activity [55]. Calcium sensing receptors The calcium-sensing receptor belongs to family C of GPCRs and can (among others) activate PLCb (Gq/11-protein) and inhibit AC (Gi/o-protein). Activation of PLCb leads to phosphatidylinositol cleavage, an increase in the intracellular calcium concentration and phosphorylation of PKC which then activates the MAPK. Activation of Gi/o-protein and adenylate cyclase inhibition result in decreased cAMP level and reduced PKA activity. The bg-subunit of the Gi/o-protein may activate Ras followed by MAPK activation and ERK1/2 phosphorylation [21,56]. Selectivity of several ligands with respect to three signalling pathways of the calcium sensing receptor has been tested – Gq/11- and Gi/o-proteins as well as ERK1/2 pathway activation. It has been found that barium and spermine exhibited efficacy biased towards Gi/o-pathway, while some polyamines and aminoglycosides were more potent in calcium-receptor mediated ERK1/2 activation [21]. 5-HT1A receptors The heptahelical, serotonin 1 receptors (A, B, D, E and F) classically couple mainly to PTX-sensitive G proteins such as Gi/o that inhibit AC-PKA signalling cascade. Among these subtypes, the 5-HT1A receptors were first to be cloned [57,58]. The 5-HT1A receptors are widely studied because of the role in regulation of mood, anxiety and cognition [15]. During coupling of 5-HT1A receptor to G-proteins, the G protein bg complex is released, which can then activate multiple effector molecules or pathways, such as the PLCb ! ERK1/2 pathway or the PI3K pathway [16,59– 61]. Phosphorylation of proteins by ERK in neurons results in receptor and ion channel activation, gene expression, and neuroplasticity, all of which may alter behaviours. The behavioural effects of the MEK/ERK signalling pathway have been reported in several studies, with MEK inhibitors causing diverse behavioural

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changes in animals, ranging from hyperactivity, reduced or increased anxiety, and depressive-like behaviour. MEK inhibitors also block the behavioural effect of antidepressants. The diverse effects of MEK inhibitors may be due to the multiple regulators and substrates linked to MEK/ERK, and to the different behavioural effects of this signalling pathway (for a review see [15]). It has been shown that 5-HT1A receptors may regulate mood-related behaviour particularly those related to depression. Nevertheless, inconsistent pharmacological, post-mortem, PET and genetic studies can be found in the literature, illustrating the complexity of the 5-HT1A receptor signalling system (for a review see [62]). It was postulated that drugs exhibiting 5-HT1A receptor functional selectivity – i.e., in different way activating various biochemical pathways would be expected to exert brain region-specific activity on serotonergic neurotransmission and possess a favourable pharmacological profile [63]. Recently, two novel 5-HT1A receptor agonists F15599 [63] and F13714 [65] were identified. F15599 is a highly selective and effective 5-HT1A receptor agonist. Neurochemical, electrophysiological and behavioural data indicate that this compound preferentially activates postsynaptic 5-HT1A receptors in rat frontal cortex [66]. F15599 influences frontal cortex pyramidal neuron electrical activity at doses that are an order of magnitude lower than those that inhibit raphe neuron electrical activity [67]. The preferential activation of post-synaptic 5-HT1A receptors by F15599 is accompanied by a remarkable capacity (as compared to F13714 and 8-OH-DPAT) to reverse phencyclidine (non-competitive antagonist of NMDA-glutamatergic receptors) induced memory/cognition deficits [68]. F15599 exhibited a potent activity, similar to that of F13714, in the forced swim test and in conditioned stress-induced ultrasonic vocalization in rats, despite the fact that the latter has an in vitro potency two orders of magnitude grater. In contrast, F15599 has a lower propensity than F13714 to induce other serotonergic signs [64,69]. F15599, both in vitro and ex vivo, exhibited different interaction and signal transduction profile from those observed for its chemical congener, F13714, and the more active (+) enantiomer of the prototypical 5-HT1A receptor agonist, 8-OH-DPAT. Thus, F15599 maximally increased G-protein activation (Emax = 102%) relative to 5-HT. Maximal efficacy was also observed for inhibition of cAMP formation in the same cell line. However, in all cases, the potency of F15599 was one to two orders of magnitude less than that of F13714 and (+)-8-OH-DPAT. In CHO-h5-HT1A cells, F15599 effectively stimulated ERK1/2 phosphorylation and, in HEK293h5-HT1A cells h5-HT1A receptor internalization. F15599 exhibited a distinctive order of potency for these responses: the pEC50 for ERK1/2 phosphorylation (7.81) was greater than that for Gprotein activation (6.97), which was similar to that for internalization (6.80), which in turn was greater than that for inhibition of cAMP formation (6.46). (+)-8-OH-DPAT also showed the highest potency for ERK1/2 phosphorylation (8.74), but the lowest potency for G-protein activation (7.17). In contrast, F13714 showed similar potency for ERK1/2 phosphorylation, internalization and G-protein activation (9.02–9.06). 5-HT showed the highest potency for G-protein activation and inhibition of cAMP formation (7.32 and 7.45, respectively), with lower potency for ERK1/2 phosphorylation and receptor internalization (6.86 and 6.80, respectively). Hence, the order of potencies for various signalling pathways was found to be different for each of the agonists tested. It was thus suggested that distinct signalling profiles and functional selectivity for specific receptor activation responses of F15599 (Table 1) underlined its favourable profile in models of cognitive and mood deficits [14]. It seems, however, not easy to identify activity relationship responsible for the preferred pharmacological properties of F15599.

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Table 1 The potency (pEC50) of F15599, F13714, (+)-8-OHDPAT and 5-HT on signal transduction responses for total G protein activation, cAMP inhibition, ERK1/2 phosphorylation and receptor internalization in cell lines stably expressing h5-HT1A receptors [63]. Efficacy model

F15599

F13714

(+)-8-OHDPAT

5-HT

Total G protein cAMP inhibition ERK1/2 phosphorylation Receptor internalization

6.97  0.08 6.46  0.10 7.81  0.13 6.80  0.04

9.02  0.12 8.67  0.02 9.07  0.11 19.06  0.16

7.17  0.05 7.77  0.38 8.74  0.06 7.38  0.02

7.32  0.05 7.45  0.18 6.86  0.09 6.80  0.12

Dopamine receptors D2 dopamine receptors (D2R) are involved in mental illnesses, an effect that was originally thought to occur through Gai/Gaomediated inhibition of AC [70]. In fact, a number of antagonists of the D2R have been recently classified as inverse agonists (by the use of a [35S]GTPgS binding assay) [71]. Recent behavioural and biochemical evidence has demonstrated that b-arrestin plays a

crucial role in signal transduction by D2R through regulation of the AKT-GSK3 pathway [72]. Stimulation of D2 receptors results in the formation of a protein complex comprising of b-arrestin 2, AKT and PP2A, which facilitates the dephosphorylation of AKT in response to dopamine [73]. This complex is a target of lithium – a drug used for the treatment of bipolar disorder and other psychiatric illnesses – the behavioural effect of which is lost in b-arrestin 2 knockout mice [74]. b-Arrestin 2 knockout mice also display a number of

Fig. 6. Major cellular signalling pathways activated by m-opioid receptor agonists (A) and the in vivo consequences of the m-opioid receptor agonists administration to a patient (B) (reprinted with permission from Ref. [83]).

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defects in different behaviour regulated by dopamine, including reduced apomorphine-induced climbing and reduced responsiveness to dopamine-dependent actions of amphetamine and morphine [73,75]. In addition, these knockout mice have a reduction in the typical novelty-induced locomotor hyperactivity phenotype of dopamine transporter knockout mice [76]. Aripiprazole (OPC-14597), an FDA-approved atypical antipsychotic drug, was one of the first functionally selective D2R ligands identified [77–80]. With the exception of aripiprazole, all FDAapproved typical and atypical antipsychotics (e.g., haloperidol, chlorpromazine, clozapine, and risperidone) share the common property of antagonizing D2-mediated G protein-dependent and independent signalling [81]. The antagonism of both signalling pathways is thought to underlie the therapeutic antipsychotic efficacy but can also cause serious extrapyramidal side effects including catalepsy and other motor dyskinesias [82]. Although aripiprazole was initially described as a partial D2R agonist, on the basis of assays performed in whole animals and isolated tissues, it was later demonstrated that it could behave as a full agonist, a partial agonist, or an antagonist depending on the signalling readout and the cell type. UNC9975, UNC0006, and UNC9994 – analogues of aripiprazole – were identified as unprecedented barrestin-biased D2R ligands. Significantly, UNC9975, UNC0006, and UNC9994 were simultaneously antagonists of Gi-regulated cAMP production and partial agonists for G protein independent D2R/barrestin-2 interactions. Importantly, in vivo studies in inbred C57BL/6 mice showed that UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects. Genetic deletion of b-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity in wild-type mice displayed by the extremely b-arrestin-biased D2R agonist UNC9994 was completely abolished in b-arrestin-2 knockout mice. Thus, it was suggested that b-arrestin signalling and recruitment could be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects [77].

m-Opioid receptors Apart from G protein mediating responses m-opioid receptors are known to interact with and regulate the function of a large number of signalling proteins such as elements of the MAPK pathway (Fig. 6). Major cellular signalling pathways activated by m-opioid receptor agonists are G protein (K+ current activation, Ca2+ current inhibition, adenylate cyclase inhibition, MAP kinases) and arrestin (MAP kinases, Scr kinase, E3 ligase, AP-2 clathrin) dependant. While the G protein-dependent signalling pathways have been established over many years, the arrestin-dependent signalling pathways have only recently begun to be characterized. The MAPK cascade can be activated via G protein- or arrestindependent pathways. Many of these pathways also lead to changes in gene expression, particularly with prolonged agonist treatments. That can be compared to the consequences of m-opioid receptor agonist administration to a patient [83]. An important question that may arise here concerns the relationship between the intracellular signalling pathways that mediate the therapeutic and adverse effects of opioid agonist. The m-opioid receptor is the target for endogenous enkephalin peptides, as well as for exogenous opioid analgesics (that are agonists) such as morphine. In addition, m-opioid receptor antagonists, such as naloxone and its derivatives, are used in the treatment of substance abuse. Enkephalins are balanced agonists for G protein- and b-arrestin-mediated activities, whereas morphine provokes considerably less receptor phosphorylation

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and internalization, which is consistent with bias towards G protein-mediated signalling [84]. However, in b-arrestin 2 knockout mice, morphine-induced analgesia is amplified and prolonged relative to wild-type mice, which is consistent with the presence of some morphine-induced b-arrestin-mediated desensitization [85]. Surprisingly, loss of b-arrestin 2 has no effect on tolerance induced by balanced agonists that strongly recruit barrestin 2, such as fentanyl and methadone [55,86]. Although the role of b-arrestin 2 recruitment and signalling in the development of opioid tolerance is still unclear, b-arrestin 2 knockout mice are protected from the side effects of morphine, including respiratory depression and constipation, suggesting that b-arrestin-mediated pathways control these peripheral side effects [87]. Therefore, a purely G protein-biased agonist may be expected to have the antinociceptive effects of the opioid analgesics without some of their problematic side effects [18].

Conclusions The simple concept of efficacy as a measure of the power of an agonist to produce cellular activation through a 7TM receptor has undergone radical modification over the past 20 to 25 years. These changes have both opened new therapeutic horizons for 7TMR drugs and also made the process of discovering and developing these new therapies more challenging. In principle one might expect from a biased agonist to activate a signalling pathway that leads to the desired clinical response but would not activate a signalling pathway responsible for adverse effects whereas an unbiased agonist would activate both pathways and produce both desired and adverse effects. Many commercially available drugs demonstrate various undesirable side effects, along with their beneficial therapeutics. Further studies of inverse agonist induced signalling might facilitate our understanding of the side effects of current drugs and take us to new avenues to efficiently design pathway-specific medications [33]. For example, for conditions such as asthma or catecholamineresistant shock, b-adrenoceptor agonists that have the capacity to signal through G proteins without desensitization mediated by b-arrestins would be predicted to be more effective than current b-adrenoceptor agonists that display significant tachyphylaxis [20,88]. The ability to visualize the multiple behaviours of 7TM receptors has shown that drugs can have many efficacies and also that the transduction of drug stimulus to various cellular stimulus-response cascades can be biased towards some but not all pathways. Thus, a diverse array of ligands can potentially be developed for a given 7TMR, each of which may have unique signalling properties. However, biased agonists potency becomes cell type dependent with the loss of the monotonic behaviour of stimulus-response mechanisms, leading to potential problems in agonist quantification. This has an extremely important effect on the discovery process for new agonists since it now cannot be assumed that a given screening or lead optimization assay will correctly predict therapeutic behaviour. Simple cell-based potency ratios may not be sufficient for prediction of therapeutic agonism in all systems. The potential therapeutic superiority of biased over unbiased ligands remains to be demonstrated in clinically relevant systems [89]. At present, biased ligands are proposed to be useful in several diseases, including heart failure, hyperlipidemia, hypertension, small-cell lung cancer, some neuropsychiatric and/or neurodegenerative disorders, hypothyroidism, osteoporosis, diabetes and Parkinson’s disease [reviewed in 28]. It will thus be interesting to see the therapeutic phenotypic profile of these and other biased molecules in future drug therapy.

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Ligand-directed trafficking of receptor stimulus.

GPCRs are seven transmembrane-spanning receptors that convey specific extracellular stimuli to intracellular signalling. They represent the largest fa...
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