The dynamic role of regulator of G-protein signalling (RGS) proteins in partial agonism

G-protein-coupled receptors (GPCRs) are important targets of pharmaceutical research. A subclass of GPCR ligands act as partial agonists, meaning that they elicit a submaximal response as compared to a full agonist. Differences in the regulation of heterotrimeric G-proteins are thought to underlie the lower activation efficacy of partial agonists. These heterotrimeric G-proteins are reciprocally regulated by guanine nucleotide exchange factors (GEFs) and a family of GTPase-activating proteins known as regulators of G-protein signalling (RGS). Although differences in GEF efficiency have been shown to underlie the submaximal response of certain partial agonists, the role of RGS proteins in this mechanism has not been assessed.

In a recent issue of The Journal of Physiology, Chen et al. (2014) investigated the role of RGS4 in partial agonism of the muscarinic type 2 acetylcholine receptor (M2R). The authors showed that a fully functional RGS4 protein, with proper localization to the plasma membrane, is necessary for partial agonism by pilocarpine and other M2R partial agonists. Importantly, this study demonstrates a novel mechanism mediating partial agonism: enhanced RGS-mediated inhibition of G-protein signalling.

Chen et al. assessed M2R signalling by measurement of G-protein-gated inwardly rectifying K⁺ (K_G) currents. The submaximal response of several M2R partial agonists relative to the full agonist, acetylcholine (ACh), was confirmed in atrial myocytes and was reconstituted in Xenopus oocytes. However, the K_G current evoked by pilocarpine and other partial agonists in oocytes lacking RGS4 was similar to that evoked by ACh, suggesting that RGS4-mediated inhibition of G-protein signalling plays an essential role in producing the submaximal effect of M2R partial agonists. In order to more definitively test this mechanism, future studies should co-express RGS-insensitive Gα subunits in Xenopus oocytes, which would be hypothesized to reduce the difference in response between M2R partial agonists and ACh.

The addition of a non-hydrolysable analogue of GTP, GTPγS, to atrial myocytes similarly reduced the difference in K_G current evoked by pilocarpine relative to ACh, implicating GTP hydrolysis, and possibly RGS4, in the mechanism underlying pilocarpine partial agonism. It was demonstrated that RGS4 did not affect the affinity of pilocarpine, as shown by concentration–response curves. Furthermore, the relative difference in response between pilocarpine and ACh was shown to be larger at more hyperpolarized membrane potentials, demonstrating that the relative efficacy of pilocarpine is voltage dependent. However, this effect was not observed in the absence of RGS4, suggesting that the voltage dependency relies on RGS4 activity.

Chen et al. (2014) then demonstrated that an intact RGS domain, in the absence of flanking amino- and carboxyl-terminal sequences, is both necessary and sufficient to mediate the partial agonism of pilocarpine. The capability of an RGS domain to mimic the action of a full RGS4 protein raises an interesting question: are the regions outside of the RGS domain actually required for mediating RGS4's effects on partial agonism? On the surface, these data appear to contradict a previous study by Srinivasa et al. (1998), which demonstrated that the N-terminal sequences are required for proper plasma membrane targeting and function of RGS4 in Saccharomyces cerevisiae. This discrepancy may be due to differences in the experimental cell model used and further work will need to be carried out to demonstrate the ability of an RGS domain to target the plasma membrane in the oocyte system.

Interestingly, Chen et al. (2014) also assessed the requirement of functional coupling of RGS4 to the plasma membrane for its effects on partial agonism. The disruption of plasma targeting of RGS4 using 2-bromopalmitate (2-BP) was shown to reduce the difference in K_G current induced by pilocarpine relative to ACh. It should be noted that 2-BP disrupts plasma membrane targeting through irreversible inhibition of palmitoyl transferases. However, this global inhibition of palmitoylation may affect various other proteins and indirectly alter the partial agonism of pilocarpine. A more reliable test of the importance of membrane targeting of RGS4 for partial agonism would be to mutate the Cys-12 residue of the N-terminus of RGS4; disruption of palmitoylation of this cysteine residue has been shown to cause RGS4 to localize primarily to the cytosol and to completely abrogate its inhibitory function of G_q protein (Bastin et al. 2012).

Finally, this study assessed whether the modulation of RGS4-mediated inhibition by pilocarpine may affect other GPCR signalling pathways. Dopamine receptor D₂ (D2R) signalling, as measured by the level of dopamine-evoked K_G currents, was shown to decrease when cells were co-treated with dopamine and pilocarpine relative to dopamine treatment alone. However, this inhibitory effect of pilocarpine on D2R signalling was absent in oocytes lacking RGS4. This suggests that pilocarpine not only produces submaximal responses on the M2R, but may also be able to inhibit other GPCR signalling pathways by promoting RGS4-mediated inhibition. It must be noted that the effect of ACh on dopamine-evoked K_G current was not assessed, and therefore full agonists may also be capable of this indirect effect on other GPCR signalling pathways.

This study effectively demonstrated the role of RGS4 in partial agonism at the M2R, but the mechanism underlying the enhanced RGS4 action is unknown. As noted by Chen et al. (2014), partial agonists induce a different conformational change in the M2R that probably results in a higher level of RGS4-mediated inhibition. Potential mediators of RGS4 modulation in the case of partial agonism are the scaffolding proteins spinophilin and its close homologue neurabin. These proteins have been shown to bind RGS4 and reciprocally modulate the level of RGS4-mediated inhibition of GPCR signalling pathways (Wang et al. 2007). The conformational changes induced by pilocarpine may cause greater recruitment of spinophilin, which could enhance RGS4-mediated inhibition of M2R signalling. Future studies should elucidate the mechanism underlying this modulation of RGS4 action; the development of pharmacological agents targeting RGS4 and its upstream modulators could enable fine tuning of GPCR signalling for therapeutic purposes.

Pilocarpine is a non-selective muscarinic partial agonist, and therefore its physiological effects may be mediated by other muscarinic receptors besides M2R. In fact, the therapeutic effects of pilocarpine are believed to be due to its action on the muscarinic type 3 receptor, which is coupled to G_q (Gabelt & Kaufman, 1992). Therefore, the physiological effects of pilocarpine may not necessarily involve the actions of RGS4 at the M2R. Determining the role of RGS4 in partial agonism at other muscarinic receptors, as well as assessing the physiological effects of pilocarpine in RGS4 wild-type and knockout mice is needed in future studies.

Moreover, although the study by Chen et al. (2014) focused on the role of RGS4, other RGS proteins (e.g. RGS5 and RGS16) may have a similar role in mediating partial agonism. The expression of other RGS proteins in the model used in this study may have recapitulated the effects of RGS4. In fact, the reduction in partial agonism by non-hydrolysable GTPγS in atrial myocytes by Chen et al. (2014) implicates any one of the RGS proteins. Consistent with our previous hypothesis, spinophilin and neurabin have also been shown to modulate the effects of other RGS proteins (Wang et al. 2007).

Relatedly, while this study focused on partial agonism of the M2R, the effects of RGS4 and other RGS proteins may also apply to a variety of GPCRs. RGS proteins are expressed throughout the body and couple to many types of GPCRs including angiotensin II receptors and μ-opioid receptors, as well as chemokine receptors. Therefore, demonstration of the role of RGS4 in M2R partial agonism by Chen et al. (2014) opens up new directions of future research aimed at identifying the roles of RGS proteins in mediating partial agonism at other GPCRs.

An interesting aspect of the results reported by Chen et al. (2014) is the ability of pilocarpine to inhibit dopamine signalling via RGS4. This indicates that pilocarpine, and possibly other partial agonists, may exert physiological effects not only by their actions on their respective receptors, but also by RGS-mediated inhibition of other GPCR signalling pathways. Moreover, this indirect inhibition may be pathway specific; therefore the effects on other pathways should be assessed in future studies. This indirect effect also suggests that if partial agonists such as pilocarpine are used in combination with other therapeutic GPCR agonists, the physiological effects of the therapy may be altered by the modulation of RGS-mediated inhibition.

Clinically, partial agonists are used to achieve a desired submaximal response and to prevent overstimulation by full agonists through competition for receptor occupancy. Given the demonstrated role of RGS4 and possibly other RGS proteins in mediating partial agonism, we can use the pattern of RGS protein expression to potentially predict the physiological effects of partial agonists. Also, changes in RGS protein expression observed in different pathophysiological conditions may alter the effects of partial agonists.

As potent inhibitors of G-protein signalling, RGS proteins can serve as excellent modulators of the level of signalling transduced by receptors. Accordingly, the study by Chen et al. (2014) provides the first demonstration of an RGS protein mediating the submaximal response of partial agonists, which may apply to a variety of other partial agonists and receptors in various tissues. Understanding the mechanism and effects of RGS protein modulation may allow us to improve treatments targeted towards GPCR signalling pathways.

Additional information

Competing interests

None to declare.

Funding

J.S. is supported by a Canadian Institutes of Health Research Master's Award. K.P.G. is supported by a Canadian Institutes of Health Research Doctoral Award – Fredrick Banting and Charles Best Canada Graduate Scholarship.