Regulators of G protein signalling (RGS) are a family of proteins that negatively regulate G protein activity by functioning as GTPase activating proteins (GAPs). 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). Here we demonstrate that stable over-expression of a myc-tagged RGS3 (RGS3myc) construct disrupts signalling via the endogenous
G_q-linked muscarinic M₃ receptor in HEK293 cells.

Human RGS3 was cloned into pcDNA3.1/myc-His (Invitrogen) to incorporate a C-terminal myc/His tag. HEK293 cells were transfected with RGS3myc DNA using Genejuice (Merck Biosciences). Transfected cells were selected using 500µg/ml Geneticin (G418). In isolated clones, stable expression of RGS3myc was confirmed by Western blotting using an anti-myc antibody (New England Biolabs). Muscarinic receptor expression was determined by binding of 1-[N-methyl-³H]scopolamine methyl chloride to intact cells to confirm expression levels similar to wild type HEK293 cells (HEK293/WT) (~40 fmol/mg protein). Signalling via the endogenous muscarinic M₃ receptor was compared between HEK293/WT and the HEK293 RGS3myc cell line (HEK293/RGS3myc). For Ca²⁺ imaging, cells were seeded onto 25mm borosilicate coverslips coated with 0.01% poly-D-lysine. Cells were loaded with 2µM fluo-3/AM and imaged on an Olympus Fluoview confocal microscope. For total inositol phosphate (InsPx) experiments, cells were seeded onto 24 well plates and loaded with 3µCi/ml [³H]-inositol for 48h.

All data are expressed as the mean ± s.e.mean for n cells unless stated. Immunohistochemistry revealed that RGS3myc was expressed homogeneously throughout the cell population with an essentially cytoplasmic localisation. In HEK293/WT a 10.97±0.41 and 12.69±0.31 (n=48) fold increase in fluo-3 fluorescence (F/F_o) was seen in response to stimulation with 1 and 100µM methacholine respectively (threshold and maximal concentrations). In HEK293/RGS3myc cells this was significantly reduced (P<0.0001; Student's t-test throughout) to 2.11±0.32 and 5.42±0.47 (n=48). Patterns of Ca²⁺ signals also varied between the cell types. In HEK293/WT cells 1µM methacholine induced repetitive Ca²⁺ oscillations that were absent in HEK293/RGS3myc cells. InsP_x generation in the presence of 10mM Li⁺ was also measured as an index of phospholipase C activity. In HEK293/WT cells a 20min stimulation with 1 or 100mM methacholine induced a 2.05±0.03 fold and 8.25±0.05 fold stimulation (n=3 experiments) over basal respectively. In HEK293/RGS3myc cells this was reduced (P<0.001) to 1.17±0.07 and 1.59±0.06 fold over basal (n=3 experiments). RGS-resistant stimulation of G subunits with aluminium fluoride (100mM NaF/10µM AlCl₃, 30min) increased InsP_x generation in both cell lines (3.1±0.3 HEK293/WT; 5.5±0.5 HEK293/RGS3myc (n=3 experiments)).

In conclusion, stable over-expression of RGS3myc leads to an alteration in the pattern and magnitude of Ca2+ signals generated via stimulation of the endogenous muscarinic M3 receptor in HEK293 cells.

Druey, K.M. et al., (1996) Nature 379, 742-745.