Single cell imaging of Ins(1,4,5)P3 production to asses RGS protein inhibition of agonist-mediated Gq signalling Stephen C Tovey & Gary B Willars. Dept. of Cell Physiology and Pharmacology, Uni. of Leicester, University Road, Leicester, LE1 9HN.

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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). The GAP activity of RGS proteins is mediated by a 120 amino acid RGS domain conserved throughout the RGS protein family. Previous studies have suggested that RGS3 acts as a GAP for both the G_i and G_q G-protein families (Druey et al., 1996). In this study we demonstrate that RGS3-mediated inhibition of G_q signalling can be measured at the single cell level using a novel Ins(1,4,5)P₃ biosensor.

Single cell Ins(1,4,5)P₃ generation was measured using cells transiently transfected with a fusion construct of eGFP and the pleckstrin homology domain of PLC-1 (eGFP-PH). This protein specifically binds to Ins(1,4,5)P₃ and PtdIns(4,5)P₂ and can be used to monitor changes in [Ins(1,4,5)P₃] in real time (Nash et al., 2001). Under resting conditions eGFP-PH is associated with the plasma membrane, but upon stimulation of G_q coupled receptors it translocates to the cytosol as PtdIns(4,5)P₂ levels fall and Ins(1,4,5)P₃ levels rise. The translocation of the eGFP signal can be followed using conventional confocal microscopy.

To examine the function of RGS3, cells were co-transfected with eGFP-PH and either an untagged or Myc epitope tagged human RGS3 construct (RGS3Myc). The expression of Myc tagged RGS3 and eGFP-PH was confirmed using Western blotting with anti-Myc (NEB, Hitchin, UK) and antiGFP (Abcam, Cambridge, UK) antibodies. All data are expressed as the mean ± s.e.mean for n cells. In HEK 293 cells stably expressing recombinant G_q-coupled muscarinic M3 receptors and transiently transfected with eGFP-PH_PLC1 a 4.3 ± 0.2 (n=82 cells) fold increase in cytosolic eGFP fluorescence (expressed as F/Fo) was seen in response to a maximal concentration of methacholine (100µM). This was reduced to 1.5 ± 0.1 (n=54 cells) and 1.7 ± 0.1 (n=71 cells) in cells co-transfected with RGS3 or RGS3Myc respectively. Cotransfection with a LacZMyc vector control had no affect (4.5 ± 0.3, n=28 cells).

The same pattern of inhibiton was seen in HEK 293 cells stably expressing the G_q-coupled catfish GnRH receptor. In cells transfected with eGFP-PH alone a 3.9 ± 0.6 (n=19 cells) fold increase in cytosolic eGFP fluorescence was seen in response to a maximal concentration of chicken II GnRH (1µM). This was reduced to 1.5 ± 0.1 (n=23 cells) and 1.8 ± 0.2 (n=16 cells) in cells cotransfected with RGS3 or RGS3Myc respectively. Once again in cells cotransfected with the LacZMyc control vector the response remained unaffected (3.5 ± 0.4, n=14 cells).

In summary, we report that RGS3 inhibition of G_q-mediated signalling via two distinct GPCRs can be assessed by imaging Ins(1,4,5)P3 production at the level of single cells.

Hepler, J.R. (1999) Trends Pharmacol. Sci. 20, 376-382.
Druey, K.M. et al., (1996) Nature 379, 742-745.
Nash, M.S. et al., (2001) Biochem J. 356, 137-142.