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First published online June 20, 2006
doi: 10.1242/10.1242/jcs.03021

Journal of Cell Science 119, 2807-2818 (2006)
Published by The Company of Biologists 2006
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Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells

R. Victor Rebois1,*,1,{ddagger}, Mélanie Robitaille2,3,*, Céline Galés2,*, Denis J. Dupré3, Alessandra Baragli3, Phan Trieu3, Nathalie Ethier3, Michel Bouvier2 and Terence E. Hébert2,3,{ddagger}

1 Laboratory of Cellular Biology, 5 Research Court, National Institute of Deafness and Communicative Disorders, National Institutes of Health, Rockville, MD 20850, USA
2 Département de Biochimie, Université de Montréal, Montréal, Québec, H3C 3J7 Canada
3 Department of Pharmacology and Therapeutics, McGill University, McIntyre Medical Sciences Building, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6, Canada


Figure 1
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  		Fig. 1. Stimulation of Kir3.2 channels by co-expressed wild-type and tagged Gß1 and G{gamma}2 subunits in Xenopus oocytes. (A) K+ currents were recorded 1-3 days after injection of cRNAs for the indicated proteins. Data for each sample represent the mean ± s.e.m. for at least five oocytes. The dashed line represents basal Kir3.2 current levels in the absence of co-expressed Gß and G{gamma} subunits. Note the lack of potentiation when Gß or G{gamma} constructs were expressed in the absence of their cognate partners. Significant increases in current were determined by an unpaired t-test (*P<0.05 compared with levels in the control), and the data is representative of at least three independent experiments. (B) Agonist-activated currents in oocytes co-expressing ß2AR. Arrows denote application and washout of 1 µM isoproterenol in KD-98 solution (see Materials and Methods). Oocytes were maintained at –80mV (a potential at which significant inward current can be measured). Data are representative of at least four separate oocytes for each experiment. (C) Data from B normalized to maximal current level to compare activation and deactivation kinetics.

 

Figure 2
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  		Fig. 2. Dose dependence of agonist-induced cAMP accumulation in cells co-expressing either AC and G{gamma}2 or AC-RLuc and GFP-G{gamma}2. HEK 293 cells were transfected with vector or with recombinant plasmids so that they expressed the indicated proteins. The cells were treated with different concentrations of isoproterenol before being assayed for cAMP levels. Dose-response curves and EC50 values were generated using GraphPad Prism® software. The EC50 values are reported as the mean ± s.d. for three independent experiments. The data presented in the graph are for a typical experiment in which the maximal agonist-induced increase in cAMP accumulation was 336 pmol cAMP/mg protein/15 minutes for HEK cells that did not express exogenous proteins (Vector) as compared with 5512 and 3070 pmol cAMP/mg protein/15 minutes, respectively for cells expressing either exogenous wild-type or tagged proteins.

 

Figure 3
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  		Fig. 3. Co-immunoprecipitation of effectors with Gß{gamma}. HEK 293 cells expressing the indicated proteins were dissolved in RIPA before immunoprecipitation. Precipitated proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose for western blotting. (A) AC-RLuc was precipitated by anti-GFP antibodies from cells co-expressing GFP-Gß1 and G{gamma}2 (left panel). No detectable RLuc was precipitated if the cells also expressed untagged Gß1 (middle panel) or if they expressed RLuc in place of AC-RLuc (right panel). (B) HA-tagged Kir3.1 was precipitated with anti-Flag antibodies from cells co-expressing Flag-tagged Gß1 and wild-type G{gamma}2. Precipitation of a HA-tagged protein required co-expression of both Kir3.1-HA and Flag-Gß1. Solid arrows represent differentially glycosylated forms of Kir3.1 as seen in native tissues (Krapivinsky et al., 1995Go). Middle panel shows cell lysates blotted for expression of HA-tagged Kir3.1. Lower panel shows immunoprecipitation of Gß by anti-Flag antibodies. Open arrow indicates specific Gß signal. The blots are representative of at least three independent experiments.

 

Figure 4
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  		Fig. 4. BRET between AC-RLuc and either GFP-G{gamma}2 or YFP-Gß1{gamma}2. (A) HEK 293 cells were transfected with recombinant plasmids to express AC-RLuc and GFP-G{gamma}2. In addition, cells expressed ß2AR, G{alpha}s and/or Gß1 as indicated. BRET was significantly increased when G{alpha}s and Gß1 were co-expressed with GFP-G{gamma}2 (**P<0.005 between groups as indicated). Isoproterenol caused a significant increase in BRET in all cells that expressed exogenous ß2AR (P<0.005). (B) HEK 293 cells were transfected to express ß2AR, G{alpha}s and AC-RLuc as well as the indicated tagged G-protein subunits (YFP1-158-Gß1, YFP159-238-G{gamma}2 and GFP-G{gamma}2) and their untagged counterpart (i.e. Gß1 or G{gamma}2). For some samples, isoproterenol caused a significant increase in BRET (*P<0.01 and **P<0.005 between groups as indicated). As a positive control, cells were transfected to express a protein consisting of YFP fused to the C-terminus of RLuc (RLuc-YFP). Transfections were performed as described in the Materials and Methods except that 0.1 µg of recombinant plasmid containing the cDNA for AC-RLuc was used per 10 cm2 tissue culture well. The data represent the mean ± s.d. for a representative experiment. (C) HEK 293 cells stably expressing the ß2AR were transiently transfected to co-express G{alpha}s, Gß1, GFP-G{gamma}2 and AC-RLuc (or CD8-RLuc as a negative control). The amount of plasmid encoding GFP-G{gamma}2 varied as represented by the ratio of fluorescence/luminescence whereas that for all the other plasmids was kept constant. Cells were treated (filled symbols) or not (open symbols) with 10 µM isoproterenol before assaying BRET. Significant differences were determined by a paired t-test for three or more experiments.

 

Figure 5
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  		Fig. 5. BRET between Kir3.1-RLuc and either GFP-Gß1, GFP-G{gamma}2 or YFP-Gß1{gamma}2. (A) BRET was measured in HEK 293 cells expressing Kir3.1-RLuc and GFP-Gß1 in the presence or absence of exogenously expressed G{gamma}2 (left) or Kir3.1-RLuc and GFP-G{gamma}2 in the presence or absence of exogenously expressed Gß1 (right). (B) BRET was measured in HEK 293 cells co-transfected with plasmids coding for Kir3.1-RLuc, GFP-Gß1, G{gamma}2 and either untagged Kir3.1 (left panel) or untagged Kir3.4 (right panel). Plasmid concentrations for the former three proteins were kept constant while those for the latter two proteins were varied as indicated in the figure (*P<0.05 and **P<0.005 versus no competitor, {phi}P<0.05 versus 1 µg competitor cDNA transfected, respectively, using a paired t-test for three or more experiments). (C) BiFC/BRET was measured in HEK 293 cells transfected to express Kir3.1-RLuc and the indicated tagged G-protein subunits (YFP1-158-Gß1, YFP159-238-G{gamma}2) together with their untagged counterparts when the split YFP-bearing partner was expressed alone. Cells co-expressing ß2AR-GFP and ß2AR-RLuc or between YFP1-158-Gß1 and YFP159-238-G{gamma}2 and CD4-RLuc served as positive and negative BRET controls, respectively and the negative controls were subtracted from raw BRET values to yield net BRET. The data represent the mean ± s.d. for five experiments. Significant differences over negative controls for ß2AR-GFP/ß2AR-RLuc or YFP1-158-Gß1 and YFP159-238-G{gamma}2/Kir3.1-RLuc were confirmed by a paired t-test (P<0.05).

 

Figure 6
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  		Fig. 6. BRET between GFP-G{gamma}2 and Kir3.1 is saturable and sensitive to receptor stimulation before surface targeting. (A) BRET saturation experiments in the ß2AR stable line co-expressing G{alpha}s, Gß1, GFP-G{gamma}2 and Kir3.1-RLuc or CD8-RLuc. The amount of plasmid encoding GFP-G{gamma}2 varied as represented by the ratio of fluorescence/luminescence whereas that for all the other plasmids was kept constant. Cells were treated (filled symbols) or not (open symbols) with 10 µM isoproterenol prior to assaying BRET. (B) Top: representative confocal image showing that Kir3.1-RLuc (green) has an intracellular distribution. Bottom: BRET experiments were performed using HEK 293 cells stably expressing the ß2AR and transiently co-expressing GFP-Gß1, G{gamma}2 and Kir3.1-RLuc. BRET was significantly increased (P<0.05) by the membrane-permeable agonist cimaterol but not by the membrane-impermeable agonist isoproterenol. Cimaterol-induced changes were blocked by pretreatment with propranolol. All ligands were used at a concentration of 1 µM. (C) Plasma membrane localization of Kir3.1 (green, middle panel) in the presence of co-expressed Flag-tagged Kir3.4 (red, top panel). Co-localization of Kir3.1 and Kir3.4 is demonstrated in the merged image (bottom panel). (D) BRET experiments were performed using the ß2AR stable line co-expressing G{alpha}s, Gß1, GFP-G{gamma}2 and Kir3.1-RLuc. Net agonist-stimulated BRET (mean ± s.d.) was significantly increased by isoproterenol (1 µM) if the cells co-expressed Kir3.4. Significant differences (*P<0.05) were determined by a paired t-test for three or more experiments in B and D.

 

Figure 7
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  		Fig. 7. BRET between tagged effectors and G{alpha} subunits is agonist-independent. HEK 293 cells were transfected to express the indicated proteins and were stimulated or not with 1 µM isoproterenol. (A) BRET was observed in cells co-expressing AC-RLuc and G{alpha}s-GFP though it was not affected by agonist stimulation, and was comparatively less than BRET between AC-RLuc and GFP-G{gamma}2. (B) BRET was also observed in cells stably expressing the ß2AR and co-expressing G{alpha}i-RLuc, Gß1, G{gamma}2 and Kir3.1-GFP. BRET was not affected by agonist regardless of whether or not Kir3.4 was co-expressed. The data represent the mean ± s.d. for at least three experiments.

 

Figure 8
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  		Fig. 8. The effects of varying protein expression levels on BRET between AC-RLuc and GFP-G{gamma}2. HEK 293 cells were transfected with recombinant plasmids to express ß2AR, G{alpha}s and Gß1, GFP-G{gamma}2 and AC-RLuc as described in the Materials and Methods so that the ratio of expressed GFP-G{gamma}2 to AC-RLuc remained constant whereas the amounts of these proteins varied over nearly two orders of magnitude (inset). Cells were left untreated (basal) or were exposed to 1 µM isoproterenol before assaying BRET in the presence of 5 µM coelenterazine H using 480 and 450-458 nm filters.

 

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© The Company of Biologists Ltd 2006