Regulators of G protein signalling (RGS) are a family of proteins that negatively regulate G protein activity by functioning as GTPase activating proteins (GAPs) (Hepler, 1999). Previous studies have suggested that RGS2, RGS3 and RGS4 act as GAPs for the G_q G-protein family (for review see Hepler, 1999). In this study we demonstrate that RGS2 and RGS3, but not RGS4 inhibit G_q-mediated signalling in HEK293 cells stably expressing recombinant muscarinic M₃ receptors (B_max 1.7pmoles/mg of protein) (HEK293/M₃ cells).

Human RGS2, RGS3 and rat RGS4 were cloned into pcDNA3.1/myc-His (Invitrogen). Expression of myc-tagged RGS2, 3 and 4 was confirmed by Western blotting using an anti-myc antibody (New England Biolabs). To examine RGS protein function, cells were transiently co-transfected with the DNA for both myc-tagged RGS and an eGFP-tagged biosensor to detect either inositol 1,4,5-trisphosphate (InsP₃ ) production (eGFP-PH) or diacylglycerol (DAG) production (eGFP-PKCC1₂). The eGFP-PH construct is the eGFP-tagged PH domain of phospholipase C1. At rest eGFP-PH is localised to the plasma membrane, but upon agonist stimulation it becomes cytoslic as InsP₃ is produced (Nash et al., 2001). The eGFP-PKCgC1₂ construct is the DAG binding domain of the C1₂ region of PKC coupled to eGFP. Under resting conditions it has a cytosolic localisation, but upon agonist stimulation and DAG production it is recruited to the plasma membrane (Oancea & Meyer, 1998). All data are the mean ± s.e.mean for n cells in 3 experiments.

In HEK293/M₃ cells transiently transfected with eGFP-PH alone a 4.2±0.5 (n=19) fold increase in cytosolic eGFP fluorescence (F/Fo) was seen in response to a maximal concentration of methacholine (100µM). This was reduced to 2.9±0.3 (n=26; P<0.05; one-way ANOVA with Dunnett's test throughout) and 1.9±0.3 (n=20; P<0.001) in cells co-transfected with either 1.5µg/well (10cm²) RGS2myc or RGS3myc respectively. Co-transfection with RGS4myc had no effect (4.4±0.3, n=19). Using 1.5mg/well of DNA the RGS3myc construct was found to express at a higher level than either the RGS2myc or RGS4myc construct. When using a matched level of expression (0.5mg/well of RGS3myc DNA), RGS3myc-mediated inhibition was similar to that of RGS2myc (2.8±0.2, n=35; P<0.05 versus control). The same pattern of inhibition was seen using the DAG sensor. In cells transfected with eGFP-PKCC1₂ alone a 3.1±0.3 (n=25) fold decrease in cytosolic eGFP fluorescence (1/(F/F_o)) was seen in response to 100µM methacholine. This was reduced to 1.5±0.1 (n=13; P<0.001) and 1.3±0.1 (n=30; P<0.001) in cells co-transfected with RGS2myc or RGS3myc respectively. Once again in cells co-transfected with RGS4myc the response was unaffected (3.0±0.4, n=20). An identical pattern of results was seen using untagged RGS proteins, although in the absence of the myc tag, expression levels could not be matched.

In summary, single cell imaging of InsP₃ and DAG production reveals that RGS2 and RGS₃ , but not RGS4 inhibit G_q-mediated muscarinic M3 signalling in HEK 293/M₃ cells.

Hepler, J.R. (1999) Trends Pharmacol. Sci. 20, 376-382.
Nash, M.S. et al., (2001) Biochem J. 356, 137-142.
Oancea, E. & Meyer, T. (1998) Cell 95, 307-318.