Here we investigate the mechanism of action for a novel class of small molecules that are effective in assays for bladder capacity and spontaneous bladder muscle contraction. We have utilized C. elegans as a model system to investigate this mechanism. Treatment of C. elegans with the compounds produced a neuromuscular phenotype, i.e., decreased egg laying, that correlated with the therapeutic activity in the small-molecule series. The phenotype was used as the basis for candidate gene analysis, and for a genetic resistance screen. Strikingly, both approaches uncovered components of the GPCR signaling pathway, a proven therapeutic target in UI disease. The genetic screen uncovered dominant mutations in two proteins (G-αq and RGS) that form an important regulatory complex.
The results of subsequent genetic and biochemical analyses were not consistent with models in which the compound acts solely on either the G-α or the RGS protein (Figure 8, models 1 and 2). However, the results are consistent with models in which the small molecule affects the RGS/G-αq complex and results in the termination of GPCR signaling, as well as alternative models involving adaptation responses to calcium signals. If the small molecule increased the affinity of the RGS protein for the G-αq transition state, there would be an increase in the rate of RGS protein GAP activity (Figure 8, model 3). Alternatively, the small molecule could stabilize RGS/G-α in an inactive complex, thus effectively acting as a noncompetitive inhibitor of the G-αq signal (Figure 8, model 4). In either case, the small molecule could interact with both the RGS protein and G-αq during the GTPase transition state. Both of these mechanisms require the physical presence of RGS protein for compound activity, and are consistent with all of the available data.
In considering these alternative models based on interaction with the RGS/G-αq complex, we were struck by reports concerning the small molecule brefeldin A and its effect on the ARF1/Sec7 complex. The relationship between ARF1 and Sec7 is partially analogous to that of G-αq and RGS: Arf1 is a small GTPase of the Ras family, while Sec7 physically interacts with Arfs to catalyze guanine nucleotide exchange (GEF activity), thereby affecting downstream signal transduction. The limitation to this analogy is that Sec7 has GEF activity whereas RGS proteins have GAP activity, thus they represent different stages of the guanine nucleotide cycle. However, in both cases it is thought that the reactions involving guanine nucleotide require formation of docking intermediates with the GTPase. Brefeldin A binds to and stabilizes the Arf1-GDP-Sec7 domain protein complex (the first intermediate on the nucleotide exchange reaction pathway). It is thought that brefeldin A prevents an important region of Arf1-GDP from reorganizing. Brefeldin A activity is also sensitive to mutations in both Sec7 and Arf1. Thus far our efforts to prove a direct effect of the compounds on an RGS/G-Protein complex have not been successful. These efforts have greatly been hampered by the difficulty in obtaining purified, active G-αq protein, and the very limited solubility of the compounds in immunoprecipitation experiments.
In addition to the models above, others remain possible. RGS proteins have been shown to interact with proteins other than G-proteins. Alternative (or additional) interaction partners include kinases, G-β5, and GPCRs. Recent unpublished data from our group suggests that these componds have a disruptive effect on an RGS protein/GPCR interaction within cells (KF, personal communication). Disruption of this interaction could conceivably result in additional free RGS. However, the significance of this disruption, and the consequences of it in the context of G-protein signaling, remains to be further investigated.
These studies have uncovered a novel set of mutants that will provide significant insight into G-α and RGS protein function. Our analysis of the compound-resistant G-α M244I allele using a yeast assay indicates that it causes increased sensitivity to continued presence of ligand (hypo-adaptation). While hyper-adaptation alleles that affect the same region of the protein have been uncovered, we believe this is the first hypo-adaptation allele to be described for G-α. Hypo-adaptation alleles of the yeast G-β protein Ste4 have been identified as suppressors of a GPA1 hyper-adaptation allele [44]. Since hypo- and hyper-adaption mutations of heterotrimeric G-protein components affect their sensitivity to GPCR agonist–antagonist activity in genetic model systems, variations of these proteins in patient populations may be related to differential responses to GPCR modifying therapies.
It is also noteworthy that we identified a dominant resistant mutation in EAT-16 (E158K) that did not affect the well-defined RGS, GGL, or DEP domains (Figure 4B). Existing crystal structures of RGS complexes are limited to the RGS domain itself, and the role of other regions of the protein is not as clear. Previous work has implicated the region of EAT-16 containing the E158K mutation in determining specificity of the RGS/G-α interaction, and in the binding and stabilizing of RGS by G-β5 [24]. It is possible that the EAT-16 E158K mutation results in an inability to bind G-β5 and consequent protein instability, leading to the observed RGS loss-of-function phenotype.
In this work, we have used an unusual combination of technologies: small-molecule screens, genetic analysis in two model systems, and biochemical assays. The relevance of the pathway and targets suggested by the model system genetics was validated by assays on mammalian systems. While the exact mechanism of action of these compounds remains under investigation, it is certain that these small molecules have a unique action downstream of muscarinic GPCRs, and that they function by limiting G-α signaling. The discovery of such compounds, as well as the unique RGS and G-α mutations uncovered here, has implications for GPCR activity modifying therapies. This work also supports the notion that small molecules affecting pathways downstream of GPCR function in novel ways, and could represent potential new therapies or biomarkers for diseases characterized by inappropriate activation of GPCR signaling.
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