Allosteric agonists represent a major advance in cholinergic pharmacology, allowing much greater selectivity for mAChR subtypes than is achievable with traditional orthosteric compounds. In the present study, we provide evidence that two structurally distinct allosteric M1 mAChR agonists effectively mobilize intracellular Ca2+ and induce phosphorylation of ERK 1/2 but are markedly different from the orthosteric agonist CCh with much less ability to rapidly recruit Arr3 and trigger compensatory mechanisms including receptor desensitization, endocytosis, and downregulation. Our finding that allosteric M1 mAChR agonists potently activate Gq-coupled signal transduction pathways while inducing minimal receptor endocytosis and degradation suggests that the specific receptor conformations stabilized by allosteric agonists may regulate distinct signaling mechanisms. This discovery has broad implications for the understanding of GPCR biology and for the application of cholinergic therapies in treating neurodegenerative and neuropsychiatric disorders.
As the understanding of GPCR signaling and regulation has been refined, much attention has focused on the role of arrestins. While originally characterized as proteins that mediate receptor desensitization and endocytosis, it is now known that arrestins can directly regulate signaling events independent of G-proteins (24
) and participate in several cell biological processes including chemotaxis (28
), stress fiber formation (29
), and protein synthesis (30
). Further investigation has shown that specific agonists for the β2
-adrenergic receptor display efficacy for arrestin-based signaling that is disproportionately higher than their efficacy for G-protein-based signaling would have predicted, leading to the coining of the term biased agonism to describe selective or preferential activation of arrestin-mediated signaling (31
). In this study, we present data demonstrating that the allosteric M1
mAChR agonists AC260584 and TBPB stimulate Ca2+
release and ERK 1/2 activation but are impaired in their ability to recruit Arr-3 following acute stimulation. We observed a more complex pattern of Arr3 recruitment following prolonged agonist treatment, with AC260584 inducing levels of Arr3 recruitment approaching levels in CCh-treated cells, whereas TBPB caused only modest Arr3 recruitment. Our results significantly extend recent studies showing that allosteric M1
mAChR agonists induce weak recruitment of arrestin 2 (β-arrestin 1) (32
). Additional studies will be required to determine the physiologic implications of allosteric mAChR agonist induced arrestin recruitment in native systems, particularly with regard to the temporal pattern of arrestin recruitment, but the present study suggests that structurally distinct ligands may signal through specific arrestin-linked mechanisms.
Previous reports have established a tight correlation between the intrinsic activity of a GPCR agonist and its efficacy for promoting receptor endocytosis (34
), providing support for the model that GPCR activation is directly linked to regulatory mechanisms that attenuate signaling and lead to receptor sequestration and downregulation. While the majority of agonists display this pattern, it has also been shown that certain GPCR agonists activate receptors without promoting receptor desensitization or endocytosis (35
), prompting revision of the model in which intrinsic activity and receptor endocytosis are fundamentally linked. Recently, Thomas et al. reported that allosteric M1
agonists related to AC260584 fail to elicit the full pattern of M1
mAChR internalization and downregulation observed with orthosteric M1
mAChR agonist treatment (36
). Here, we demonstrate that two structurally distinct allosteric agonists activate the M1
mAChR while inducing much less compensatory receptor endocytosis and downregulation than the orthosteric agonist CCh. These differential effects suggest that allosteric agonist binding may put the M1
mAChR in a conformation in which it interacts with certain intracellular signaling and/or scaffolding proteins but not others. Recently, Li and colleagues demonstrated that different classes of agonists induce distinct structural changes in the M3
mAChR subtype (37
), providing evidence for a molecular basis by which distinct agonists acting on a single receptor can differentially regulate signaling pathways. It is possible that in addition to activating signaling cascades shared by orthosteric agonists, allosteric agonists could also regulate additional pathways. Privileged signaling regulated by allosteric agonists is beginning to be explored for a variety of GPCRs including metabotropic glutamate receptors (39
), providing an intriguing and potentially clinically useful aspect of GPCR signaling.
An alternate explanation for our observation of blunted arrestin 3 recruitment and receptor endocytosis is that the allosteric agonists evaluated in this study display a lower efficacy compared to that of CCh, rather than a bias in agonism. Because radioligands for allosteric binding sites on the M1 mAChR do not exist, it is impossible to precisely determine the relationship between occupancy and efficacy for allosteric compounds using traditional methods of receptor affinity and competitive binding. However, careful analysis of the available data aid in the interpretation of this question. The potency of CCh at each of the responses measured in this study gives clear insight into the levels of receptor reserve for the individual assays. CCh has an affinity for the M1 mAChR in the low micromolar range. CCh has a potency for activating M1 coupling to ERK 1/2 phosphorylation and for activating arrestin 3 recruitment in the micromolar range. The finding that CCh potencies in these assays are the same as CCh affinity for the M1 mAChR suggests that there is no significant receptor reserve for the activation of ERK 1/2 phosphorylation or arrestin 3 recruitment. In contrast, CCh has a potency of approximately 100 nM for the activation of Ca2+ mobilization. This suggests that there is significant receptor reserve when using CCh in the Ca2+ mobilization assay. Interestingly, both AC260584 and TBPB have potencies in the 10−100 nM range for the activation of ERK 1/2 phosphorylation and arrestin 3 recruitment. If there is little or no receptor reserve in these assays (as suggested by the CCh potency), this is likely to reflect the true efficacies of these compounds in eliciting these responses.
It is also important to note that CCh, TBPB, and AC260584 all have submicromolar potencies in the Ca2+ mobilization assay. While it is clear that this assay displays significant receptor reserve when CCh is used as the agonist, the potencies of TBPB and AC260584 are similar in all three assays. This suggests that differences in receptor reserve do not have the same influence on the potencies of these allosteric agonists as they do with CCh. The fact that the potencies of TBPB and AC260584 are constant across assays suggests that the differences in efficacy of TBPB at inducing arrestin 3 recruitment are not likely to be explained simply by differences in receptor reserve in the different assays. However, because we cannot directly assess affinities, we cannot fully evaluate the occupancy/efficacy relationships for the three compounds. Therefore, we cannot definitively rule out the possibility that a more traditional view of TBPB as a partial agonist with similar efficacies across signaling pathways could explain these results.
The fact that AC260584 and TBPB do not rapidly recruit arrestin or induce M1
mAChR endocytosis may have important pharmacological and cell biological implications. Agonist-induced receptor endocytosis and lysosomal degradation could limit efficacy over extended periods of administration, making allosteric agonists that do not induce these compansatory changes attractive targets for chronic therapeutic applications. Indeed, studies in acetylcholinesterase knockout mice have revealed that the loss of this enzyme, which regulates attenuation of signaling at cholinergic synapses, results in significant downregulation of mAChRs, abberrant receptor trafficking, and blunted response to agonist stimulation (40
). These perturbations in the cholinergic system serve as a model for the alterations that likely occur following chronic administration of cholinesterase inhibitors, the predominant therapy for Alzheimer’s disease (AD), and may account for the limited clinical efficacy of these drugs. It is worth noting, however, that there is evidence supporting a role for arrestin-mediated endocytosis in maintaining the ability of a GPCR to respond to repeated agonist stimulation. Whistler et al. showed that morphine, an agonist at the μ-opioid receptor, fails to promote arrestin recruitment and receptor internalization, in contrast to the μ-opioid receptor agonist etorphine (35
). Interestingly, morphine causes more physiological tolerance and dependence than etorphine, and the authors hypothesize that persistant receptor activation in the absence of desensitization, endocytosis, and recycling triggers alternative mechanisms of compensation that lead to tolerance. The effects of chronic in vivo
administration of allosteric M1
mAChR agonists need to be investigated directly in order to determine whether they induce functional changes in vivo
following repeated administration. It is conceivable that drug discovery efforts could include arrestin recruitment as a key screening parameter for the development of future M1
Subtype-selective allosteric agonists represent a tremendous advance in cholinergic pharmacology and will likely have a major impact on cholinergic-based therapies for neurological and neuropsychiatric disorders. The findings of this study complement a growing body of literature indicating that GPCR signaling is remarkably diverse and that structurally distinct agonists differ with respect to the profiles of responses they elicit. Ongoing investigation in this exciting field should continue to enhance both the understanding of basic receptor biology and the utility of clinical pharmacotherapy.