This is illustrated in Figure 2A, where signalling pathways 1–3 show progressively lower receptor/effector coupling. Drug A is a full agonist in pathway 1, a strong partial agonist in pathway 2 and a weak partial agonist in pathway 3. Drugs B and C display the same pattern, but have lower efficacy, and act as partial agonists even in the strongly coupled pathway 1, and have weak or no agonist activity for pathways 2 and 3 (and are highly likely to be antagonists of these pathways). Thus, these three drugs have the same rank order of efficacy for separate effector pathways, and the differences in responses can be explained purely by the coupling efficiency of the receptor to each effector. In Figure 2B, this is also the case for pathways 1 and 3, but drug B now has a much higher efficacy than drug A in pathway 2. The observation that drug A has higher efficacy than drug B for pathway 1, but a lower efficacy than drug B for pathway 2 is generally referred to as a ‘reversal of efficacy’ (Urban et al., 2007). This term can be used only when comparing two different drugs across two pathways, and does not refer to the ability of a particular drug to act as an antagonist for one pathway, but an agonist for another pathway. The most likely interpretation of an observed reversal of efficacy is that the two drugs are inducing or stabilizing different receptor conformations that in turn couple preferentially to different signalling pathways (Kenakin, 2007). Thus, reversal of efficacy provides a relatively unambiguous demonstration of ligand-directed signalling, that is, it represents a sufficient condition to state that this is occurring. However, we discuss below the evidence that reversal of efficacy is not always a necessary condition to be able to identify ligand-directed signalling.
Agonists and partial agonists promote distinct β-AR conformations
As shown in Figure 2, the capacity of two or more drugs to display ligand-directed signalling can be convincingly demonstrated by the measurement of multiple downstream signalling outputs and demonstration of reversal of efficacy (Urban et al., 2007). However, it is now also possible to examine changes in receptor conformation directly. This approach has been used to show that the endogenous β-AR agonist NA, catechol itself and the partial agonist salbutamol produce distinct modes of β2-AR activation (Swaminath et al., 2005). Each element within the structure of catecholamines has been shown to interact with amino acid side chains in the ligand-binding pocket of the β2-AR (Liapakis et al., 2004; Swaminath et al., 2004). The hydroxyl groups on the catechol ring undergo hydrogen bonding with Ser203 (5.42), 204 (5.43) and 207 (5.46; Strader et al., 1989a; Liapakis et al., 2000), the chiral β-hydroxyl interacts with Asn293 (6.55; Wieland et al., 1996; Bhattacharya et al., 2008; Reynolds et al., 2009), the aromatic ring undergoes hydrophobic interaction with Phe290 (6.52; Strader et al., 1989b) and the bioamine–NH3+ group interacts with Asp113 (3.32; Strader et al., 1988). In addition, the amine substituent group present in full agonists such as adrenaline and ISO may interact with as yet unidentified residues in TM6 and TM7 (Liapakis et al., 2004; Swaminath et al., 2004). While optimal agonist efficacy is associated with the presence of all the elements present in adrenaline and ISO, compounds missing only one element, including NA, N-methyldopamine and salbutamol, are still strong partial agonists, and compounds missing two or more elements can still be weak partial agonists (Liapakis et al., 2004). It is interesting to note that the catechol analog U-0521 (3′,4′-dihydroxy-2-methylpropio-phenone), which lacks an amine group, demonstrates positive chronotropic effects in rat atria, albeit with about 105-fold lower potency than ISO (Kaumann et al., 1977). In fact, U-0521 also increases the spontaneous beating rate of isolated ventricular myocytes despite its inability to promote cAMP accumulation, or to act as an antagonist for ISO-stimulated responses. These early findings perhaps indicate that U-0521 promotes an active receptor conformation distinct from that of ISO.
Reconstituted phospholipid vesicles containing β2-ARs labelled at Cys265 (6.27) with the environmentally sensitive fluorophore tetramethylrhodamine maleimide were used to show that the receptor undergoes two kinetically distinct conformational changes upon agonist activation (Swaminath et al., 2004). It was then demonstrated that whereas NA induces a biphasic conformational change composed of both rapid and slow phases, catechol induces only the rapid phase, and salbutamol induces only the slow phase. Interestingly, catechol was still able to induce the rapid conformational change following pre-incubation of the receptor with the partial agonist salbutamol, with the antagonists timolol and alprenolol or the inverse agonist ICI118551, but not after pre-incubation with NA or ISO (Swaminath et al., 2005). All β-AR ligands with a charged amine group interact with Asp113 (3.32). Molecular modelling indicated that catechol, NA and ISO also share the TM5 binding sites in the lower part of the binding pocket, but the aromatic ring of salbutamol appears to be orientated towards the upper part of the binding pocket and extracellular loop (ECL) 2 (Swaminath et al., 2005). This orientation of salbutamol prevents interaction between the chiral β-hydroxyl and Asn293 (6.55), consistent with earlier findings that the affinity and efficacy of partial agonists that lack a catechol group are not affected by mutation of Asn293 (Wieland et al., 1996). By inference, β-AR antagonists interact with Asp113 (3.32), but other than this, each compound would be expected to produce a unique network of interactions with amino acid side chains in the binding pocket.
These β2-AR conformational studies indicate that there are not only quantitative differences in the activity of agonists and partial agonists, but also qualitative differences in their capacity to induce particular receptor conformations. Further work using live cells has confirmed that β-AR agonists display ligand-directed signalling. An elegant study has recently shown that agonist stereoisomers promote differential Gs/Gi coupling of the β2-AR in adult rat cardiomyocytes (Woo et al., 2009). Fenoterol is a β2-AR-selective partial agonist with two chiral centres, and can be synthesized as S,R; R,R; S,S; or R,S isomers. The S,R and R,R isomers of both fenoterol and the related compound methoxyfenoterol all stimulated cardiomyocyte contractility that was blocked in the presence of the β2-AR-selective inverse agonist ICI118551. Importantly, the R,R isomers had higher potency than the S,R isomers, and the S,R but not the R,R responses showed a clear leftward shift in the presence of PTX. The PTX sensitivity was even more apparent in Erk1/2 phosphorylation assays, with the (S,R)-fenoterol response largely blocked, but the (R,R)-fenoterol response unaffected. Differential Gα coupling was verified by subtype-specific immunoprecipitation of activated Gα subunits labelled with [γ-32P]GTP-azidoanilide. For example, the S,R isomer of fenoterol stimulated substantially higher activation of Gαi2 than the R,R isomer, whereas (R,R)-fenoterol produced a threefold higher activation of Gαs than (S,R)-fenoterol. These results are compatible only with the conclusion that the two stereoisomers stabilize distinct conformations of the β2-AR. This work provides a clear demonstration of ligand-directed signalling in a physiological system, and may have important clinical implications. The authors suggest that agonists that selectively stimulate β2-AR Gs coupling, without stimulating β2-AR Gi coupling or β1-AR activation, may have considerable therapeutic benefit (Woo et al., 2009).
Further demonstrations of ligand-directed signalling by β-AR agonists have used recombinant systems. Recently, 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 receptors and interacting proteins. The data obtained can then be compared with associated signalling outputs, namely changes in cAMP or Erk1/2 phosphorylation (e.g. Drake et al., 2008). One such study has demonstrated that, relative to ISO, drugs acting as partial agonists for cAMP production can nonetheless act as full agonists for arrestin-3 recruitment (Drake et al., 2008). In fact, cyclopentylbutanephrine (CPB) and ISO demonstrate the reversal of efficacy that verifies the presence of ligand-directed signalling, while α-ethylnoradrenaline and isoetharine are both partial agonists for cAMP, but full agonists for arrestin-3 recruitment. Unlike these three drugs, the remaining full or partial agonists (adrenaline, NA, methylnoradrenaline, protokylol, deoxyadrenaline, zinterol, metaproterenol, terbutaline, fenoterol, procaterol, formoterol, albuterol, salbutamol and salmeterol) all have equivalent efficacy in the two assays. It was pointed out that drugs, such as CPB, α-ethylnoradrenaline and isoetharine, which display ‘biased agonism’ towards arrestin recruitment, share an ethyl group on the αC atom (Drake et al., 2008). As this is in close proximity to the NH3+ group that interacts with Asp113 (3.32), there may be a steric effect of this α-ethyl group that compromises receptor conformational changes linked to G protein activation, without affecting those that promote phosphorylation or arrestin binding. In fact, addition of the α-ethyl group in α-ethylnoradrenaline substantially improves the rate of arrestin-3 recruitment without changing cAMP production relative to NA. It should be noted that these studies were done using HEK293 cells expressing the β2-AR at 1 pmol·mg−1 protein, and that the agonists were used at a concentration 100 times their KD, in some cases up to 300 µM (including NA and ethylnoradrenaline). While these conditions are non-physiological, the experiments have clearly been designed to ensure maximal receptor occupancy, such that the observed responses solely reflect efficacy, or maximum agonist effect values. The main drawback of using such high concentrations would be the possibility of off-target effects. This is unlikely for the arrestin recruitment studies, however, as the FRET response can only involve the expressed β2-ARs that are tagged with cyan fluorescent protein.
Another study that compared cAMP accumulation with activation of cAMP response element (CRE)-mediated reporter gene transcription provides a third demonstration that agonists and partial agonists promote qualitatively different conformations of the β2-AR (Baker et al., 2003b). While both of these responses are downstream of the receptor activation process, they differ substantially in their degree of signal amplification and assay timing (10 min for cAMP accumulation and 5 h for the reporter gene assays). Most notably, the longer time period required for CRE reporter gene transcription favours receptor desensitization more than the short-term cAMP assay. The partial agonists salbutamol and terbutaline display efficacies for CRE activation that are comparable with those of the full agonists ISO and adrenaline. However, salbutamol and terbutaline are, respectively, 16 and 19 times more potent in the reporter gene assay than for short-term cAMP accumulation, whereas ISO and adrenaline are four- and sixfold less potent in the reporter gene assay compared to cAMP. The antagonists ICI118551 and propranolol show a two- to sixfold lower affinity for the β2-AR based on their capacity to block ISO or adrenaline-stimulated CRE activation compared to their antagonism of cAMP responses. Furthermore, based on the CRE responses, ICI118551 and propranolol show a 10-fold lower affinity at the β2-AR in the presence of ISO or adrenaline compared to salbutamol or terbutaline. This discrepancy in antagonist pKB values was not seen in the cAMP assays. The reporter gene assay data are not consistent with the generally held view that antagonist affinity is constant for a particular receptor, irrespective of the agonist or the bioassay used. Indeed, they suggest that the properties of the receptor are altered depending on whether it is activated by a full or a partial agonist. This alteration can be attributed in part to previous findings that the β2-AR is phosphorylated to a greater extent in response to full compared to partial agonists (January et al., 1997). When the reporter gene assays were carried out in cells expressing a mutant β2-AR lacking all of the possible PKA and GRK phosphorylation sites, there were two important differences in the data obtained. Firstly, ISO was substantially more potent at the mutant β2-AR (pEC50 9.45 vs. 8.11 for the wild-type receptor), and secondly, there was only a twofold difference in the pKB values obtained for ICI118551 between ISO and salbutamol, compared to the 10-fold difference seen with the wild-type β2-AR (Baker et al., 2003b). This study brings together various previous findings and new ideas, namely that the receptor conformations induced by the full agonist ISO and the partial agonist salbutamol differ in their capacity for phosphorylation and desensitization of responses, and that the conformation of the β2-AR that is phosphorylated and possibly interacting with additional proteins has a lower affinity for antagonists than the non-phosphorylated state.
β-AR ligands that are antagonists for cAMP accumulation are able to activate MAPK phosphorylation
The concept of ligand-directed signalling is a topic of immense interest and has recently been extended to drugs that act as antagonists for the cAMP pathway. As stated by Urban et al. (2007), ‘At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor’. Several studies describe the activation of Erk1/2 phosphorylation by drugs classified as β-AR antagonists in cells expressing β1- or β2-AR (Azzi et al., 2003; Baker et al., 2003a). It has now been demonstrated that a wide range of β-AR ligands have complex efficacy profiles for cAMP generation and Erk1/2 activation at both β1- and β2-ARs (Galandrin and Bouvier, 2006). In addition, recent studies on mouse (Sato et al., 2007) and human (Sato et al., 2008) β3-ARs showed that the antagonists SR59230A and L748337 act as classical competitive antagonists for cAMP accumulation, but are powerful agonists for both Erk1/2 and p38 MAPK activation. These effects again suggest that many compounds previously thought to interact with receptors to block the actions of agonists (as antagonists or inverse agonists) may in fact have the ability to selectively activate discrete pathways by inducing or interacting with particular conformations of the receptor. The idea that compounds acting as antagonists can in fact induce an active receptor conformation is not novel, as it has been known for some time that non-conventional partial agonists at high concentrations have cardiostimulant effects and produce cAMP accumulation via the β1-AR (reviewed by Kaumann & Molenaar, 2008). For example, the compound CGP12177A blocks agonist-stimulated cAMP accumulation at low concentrations, but also binds to a low-affinity ‘agonist site’ utilizing interactions with residues that are distinct from the high-affinity ‘catecholamine site’ of the β1-AR (Joseph et al., 2004; Baker et al., 2008).
The fact that many compounds previously regarded as ‘blockers’ express their own spectrum of pharmacological properties has potentially far-reaching consequences for the use of these drugs therapeutically. To date, there is not extensive literature that directly relates clinical efficacy to the ability of β-AR antagonists to act as agonists for MAPK or other signalling pathways; however, this area is likely to expand greatly in the near future. It has been suggested that the therapeutic benefits of carvedilol in heart failure patients are related to its unique capacity to activate Erk1/2 signalling by a G protein-independent mechanism (Wisler et al., 2007), but it is difficult to draw conclusions on the basis of one compound given that other β-blockers have similar clinical efficacy. To highlight an approach based on a series of different compounds, a key example of functional ligand selectivity is the finding that antipsychotic drugs acting at the dopamine D2 receptor can have opposite effects on Gi/o-mediated decreases in cyclic AMP compared to receptor recruitment of arrestin-3 (Masri et al., 2008). All clinically effective antipsychotics block arrestin-3 recruitment, despite having effects on Gi/o coupling that vary widely, ranging from partial agonists to neutral antagonists and inverse agonists. Here again, further work is needed to determine the downstream signalling pathways that are inhibited by antipsychotics, although the authors suggest that Akt and GSK-3 are important effectors.
Recent studies provide insights into how antagonists activate MAPK signalling. An array of β-AR blocking agents have been tested for their capacity to stimulate cAMP production (using the ICUE2 sensor) or Erk1/2 phosphorylation in HEK293 cells stably expressing the β2-AR (Wisler et al., 2007). β-AR blocking agents that are partial agonists for cAMP accumulation, namely acebutolol, alprenolol, atenolol, labetalol, oxprenolol, pindolol and practolol, also stimulated Erk1/2 phosphorylation. All of the other agents tested (betaxolol, bisoprolol, carvedilol, ICI 118551, metoprolol, nadolol, propranolol, sotalol and timolol) are inverse agonists for cAMP in cells pretreated with 250 µM IBMX. Of these, only carvedilol and propranolol stimulated Erk1/2 phosphorylation. To determine the mode by which these two drugs promote Erk1/2 signalling, a mutant β2-AR was used that is deficient in G protein activation (T68F,Y132G,Y219A) (Wisler et al., 2007). Relative to ISO, carvedilol still acted as a partial agonist for Erk1/2 phosphorylation, whereas propranolol produced no response. Previous experiments had shown that ISO produces its response partly by coupling to Gi, and partly by receptor phosphorylation and recruitment of arrestin (Shenoy et al., 2006). Not surprisingly, the carvedilol Erk1/2 response was sensitive to depletion of arrestin-3 by siRNA, but was not sensitive to PTX. Thus, carvedilol, but not propranolol, causes receptor phosphorylation, recruitment of arrestin3–GFP and receptor internalization without changes in cAMP levels (Wisler et al., 2007). This study indicates that carvedilol induces or stabilizes a β2-AR conformation that does not activate G proteins, but can facilitate activation of arrestin-dependent signalling.
A similar study carried out in HEK293 cells expressing the human β1-AR showed that ISO stimulates Erk1/2 phosphorylation by both Gi-dependent and G protein-independent pathways (Galandrin et al., 2008). Relative to ISO, bucindolol was a partial agonist and propranolol an inverse agonist for cAMP, whereas both bucindolol and propranolol stimulated Erk1/2 phosphorylation (Emax ∼ 30% of ISO response). The ISO-stimulated Erk1/2 response was partially blocked by PTX (30% of control) and by βARK-CT (a C-terminal peptide derived from GRK2 that sequesters Gβγ subunits), but the bucindolol and propranolol responses were unchanged (Galandrin et al., 2008). In cells co-expressing Gαi1 tagged with Renilla luciferase (Gαi1-91hRluc), Gγ2 tagged with green fluorescent protein (GFP10-Gγ2) and untagged β1-AR, only ISO caused a reduced BRET signal due to dissociation of Gα and Gγ subunits. BRET was also measured in cells co-expressing β1-AR-hRluc and GFP10-Gγ2 in the presence of untagged Gαi1, or alternatively β1-AR–GFP10 and Gαi1-91hRluc. In both cases, the conformational change induced by ISO (10 µM) caused an increase, whereas bucindolol and propranolol decreased the BRET signal. These experiments show that ISO and two prototypical antagonists bucindolol and propranolol promote distinct conformations of the β1-AR.
Whereas it is clear that activation of Erk1/2 phosphorylation by carvedilol at the β2-AR involves arrestin recruitment (Wisler et al., 2007), the β1-AR responses to ISO, bucindolol or propranolol were not sensitive to co-expression of a dominant negative arrestin-2 or siRNAs that knock down arrestin-2/3 (Galandrin et al., 2008). Instead, the c-Src inhibitor PP2 caused almost complete blockade of Erk1/2 phosphorylation for all three ligands, suggesting that the upstream signalling pathways activated by these ligands converge at or above the level of c-Src tyrosine kinases. This raises an interesting point, as like the β3-AR, there are Pro-X-X-Pro motifs in the third intracellular loop and the C-terminal tail of the β1-AR that could conceivably mediate interaction with c-Src or other SH3 domain proteins. It has been shown in vitro that the β1-AR third intracellular loop does not bind c-Src directly. The entire loop containing the proline-rich motif binds specifically to endophilins (SH3p4/p8/p13). but not to other SH3 proteins including the adapter protein Grb2, c-Src or the synaptic vesicle trafficking protein amphiphysin 2 (Tang et al., 1999). It is possible that the C-terminal tail of the β1-AR does bind c-Src, or alternatively, that interaction between this receptor and c-Src is mediated by adapter proteins other than arrestins. We suggest that binding of bucindolol or propranolol to the β1-AR promotes a conformation that is able to activate c-Src without G protein involvement, while ISO promotes a conformation that can activate both Gi and c-Src, or alternatively, conformations that can activate each of these pathways independently.
The β2-AR lacks any Pro-X-X-Pro motifs, but there is evidence that c-Src can bind directly to helix 8 (Sun et al., 2007). Despite this, the β2-AR is more commonly found to activate c-Src by direct Gαs interactions (Ma et al., 2000), direct Gαi interactions (Ciccarelli et al., 2007), PKA phosphorylation (Schmitt and Stork, 2002b) or arrestin recruitment (Luttrell et al., 1999). The β1-AR findings described earlier indicate that Erk1/2 phosphorylation in response to ISO, bucindolol or propranolol does not involve arrestins (Galandrin et al., 2008). Another recent study suggests, however, that carvedilol and alprenolol act at the β1-AR to promote arrestin-2/3 recruitment and consequent transactivation of the EGF receptor and Erk1/2 phosphorylation (Kim et al., 2008). In agreement with this, propranolol was unable to stimulate arrestin recruitment, and bucindolol was not tested (Galandrin et al., 2008). These studies provide strong evidence that different drugs may have distinct modes of action not only with respect to cAMP and Erk1/2 signalling, but also in terms of the upstream signalling effectors that they activate. There is one caveat, as the study describing the actions of carvedilol and alprenolol used the mouse β1-AR, while the effects of propranolol and bucindolol were demonstrated in cells expressing human β1-ARs (Galandrin et al., 2008; Kim et al., 2008). Although both the human and mouse β1-AR have multiple Pro-X-X-Pro motifs in the third intracellular loop and the C-terminal tail, there may be other amino acid differences that differentially affect phosphorylation or arrestin recruitment.
At the β3-AR, drugs that act as antagonists of cAMP responses strongly activate Erk1/2. However, the Erk1/2 responses do not involve phosphorylation of the receptor, interaction with arrestins or internalization because the β3-AR does not undergo any of these processes. In CHO-K1 cells expressing the human β3-AR at physiological levels, the β3-AR ligand L748337 is a competitive antagonist for cAMP accumulation, but has high agonist potency and efficacy for Erk1/2 phosphorylation. Zinterol, on the other hand, which has agonist properties at the human β3-AR (Hutchinson et al., 2006), had high efficacy for cAMP accumulation, but lower efficacy than L748337 for both Erk1/2 and p38MAPK phosphorylation (Sato et al., 2008). Reversal of efficacy was also demonstrated between the agonist CL316243 and the antagonist SR59230A acting at the mouse β3-AR (Sato et al., 2007). When the functional readout is cAMP, CL316243 is a full agonist and SR59230A either a partial agonist or antagonist depending on the level of receptor expression. In the identical cells but using extracellular acidification rate (ECAR) as the functional measure, both CL316243 and SR59230A are full agonists at all levels of receptor expression. Further analysis using selective MAPK inhibitors and Western blotting confirmed that SR59230A has much higher efficacy than CL316243 for MAPK signalling. These examples of reversal of efficacy provide strong support for the concept of ligand-directed signalling.
In addition, there is evidence from the studies with β3-AR that MAPK responses induced by agonist ligands differ from those induced by antagonist ligands in terms of the G proteins utilized. Thus, L748337 stimulation of the human β3-AR causes an Erk1/2 response that is largely blocked by PTX, indicating that the antagonist recognizes or induces a conformation of the β3-AR that efficiently couples to Gi/o but not to Gs. Indeed, in the presence of a classical agonist, L748337 blocks the capacity of the β3-AR to adopt a conformation capable of Gs coupling (Sato et al., 2008; Skeberdis et al., 2008; Wuest et al., 2009). The Erk1/2 response to the agonist ligand L755507 is much less affected by PTX, suggesting coupling predominantly to Gs (Sato et al., 2008). This finding again highlights differences between the three human β-ARs, as antagonist-stimulated Erk1/2 phosphorylation at the β1- and β2-AR is not PTX sensitive (Wisler et al., 2007; Galandrin et al., 2008).
Studies of ligand-directed signalling in CHO-K1 cells expressing the mouse β3-AR demonstrate additional complexity for the interpretation of efficacy data (Sato et al., 2007). In cells with high β3-AR expression levels (950 fmol·mg−1 membrane protein), SR59230A caused increases in ECAR measured in the cytosensor microphysiometer, and behaved as a weak partial agonist for cAMP accumulation. In 3T3-F442A cells that express endogenous β3-ARs or in CHO-K1 cells expressing low levels of the β3-AR (115 fmol·mg−1 protein), SR59230A still produced full cytosensor responses, but no measurable cAMP accumulation. Changes in ECAR in the low-expressing cells were abolished by the p38MAPK inhibitor RWJ67657, adding to the evidence that SR59230A caused robust phosphorylation of p38MAPK. In fact, the efficacy of SR59230A for the p38 MAPK response in the low-expressing cells was greater than in the cells with high β3-AR expression. A clue to the cause of this surprising finding came from the observation that the efficacy of CL316243 was only one-third of that displayed by SR59230A in low-expressing cells, and in high-expressing cells CL316243 failed to produce a measurable p38MAPK response (Sato et al., 2007). Because p38MAPK phosphorylation was inhibited by 8-Br-cAMP, we concluded that p38MAPK activation is attenuated by cAMP, a situation that occurs to a greater extent in high-expressing cells. While in many cases there is positive cross-talk between signalling pathways, the study demonstrates a negative interaction between p38MAPK and cAMP in CHO-K1 cells expressing mouse β3-AR.
Studies done at the level of receptor conformational changes; interactions between receptors and G proteins or alternative effectors, such as arrestins; or G protein activation are likely to provide a clear indication of the capacity of ligands to stabilize or induce distinct receptor conformations. It is also worthwhile to study ligand-directed signalling based on downstream signalling events, as these are of potential importance in optimizing the clinical efficacy of new or existing drugs. Our studies on the β3-AR indicate, however, that measuring downstream signalling events can cloud the interpretation of ligand-stimulated responses, as there may be composite effects of conformational bias at the level of the receptor, plus signalling pathway interactions that produce either synergy or essentially ‘functional antagonism’, as in the case of p38 MAPK and cAMP (Sato et al., 2007). Our findings sound a cautionary note regarding the use of recombinant cell systems with extremely high levels of receptor expression for studies of pleiotropic signalling by β-AR antagonists, as the likelihood of pathway interactions may be greatly increased.
The studies described above use cAMP and ECAR as functional readouts to identify ligand-directed signalling. ECAR is a useful screening technique that identifies changes in metabolic activity in cells, and therefore, makes no assumptions regarding the pathways being activated. We have also used reporter genes in studies of human β3-ARs to map the efficacy of different agonists and antagonists for a range of signalling pathways. The data have been used to generate a ‘web of efficacy’ (Figure 3) that compares the efficacy of ligands in four reporter gene assays, and provides a profile for the series of β-AR ligands tested. Although at a comparatively early stage, it is interesting to note that carvedilol and nebivolol display similar profiles and quite different from that shown by the prototypical β-AR antagonist propranolol. In contrast, bupranolol behaved as a neutral antagonist in all of the reporter gene assays tested to date. The human β3-AR-selective ligands L755507 (agonist) and L748337 (antagonist) affect a similar spectrum of reporter genes, but with different efficacy. Hopefully, this approach will provide another avenue for the identification of ligand-directed signalling.
Figure 3. The web of efficacy. A series of seven β-AR ligands were compared using four reporter gene assays, activator protein-1 (AP-1; JNK); cAMP response element (CRE; PKA and JNK/p38MAPK); nuclear factor of κB (NF-κB); and serum response element (SRE; MAPK/JNK). Note the similar profile exhibited by carvedilol and nebivolol, and the different profile shown by the prototypical β-AR antagonist propranolol. Bupranolol was a neutral or inverse agonist in the reporter gene assays. The human selective β3-AR ligands L755507 and L748337 had similar profiles apart from that in the AP-1 reporter gene assay.
Download figure to PowerPoint