# G\(\gamma\) and G\(\alpha\) Identity Dictate a G-Protein Heterotrim Plasma Membrane Targeting
## 3 Results
### Analysis of G-Protein Interactions in Live Cells Using FLIM-FRET Microscopy
approximately three times lower than for G\(\alpha\)s. These findings suggest a lower G\(\alpha\)s\({}_{\rm IEE}\) heterotrim content than that of G\(\alpha\)s at the plasma membrane, which led to a reduced activation of adenylyl cyclase. To further confirm that the observed increase in cAMP concentration over the basal level resulted from the direct interaction of G\(\beta_{1}\) with G\(\alpha\)s\({}_{\rm IEE}\), we performed a pull-down assay. Cell lysates containing  
Figure 4: Effect of different G\(\gamma\) subunits on the production of cAMP. HEK 293 cells were transiently transfected with Citrine–G\(\alpha\)\({}_{3}\), dopamine receptor D\({}_{2}\)R, mCherry–G\(\beta_{1}\gamma_{2}\)/\(\gamma_{9}\), or Citrine–G\(\alpha\)s/s\({}_{\rm IEE}\), adenosine receptor A\({}_{2\rm A}\)R and mCherry–G\(\beta_{1}\gamma_{2}\)/\(\gamma_{9}\). The activity of the investigated proteins after stimulation of the D\({}_{2}\) receptor with luminarole maleate or the A\({}_{2\rm A}\) receptor with 2-phenylaminoadenosine was determined by measurements of cAMP levels. Data are presented as percentage of mean cAMP level in control (non-transfected cells) which are considered as intrinsic cAMP levels after stimulation of the D\({}_{2}\) or A\({}_{2\rm A}\) receptor. Differences in cAMP levels between samples were evaluated by the unpaired \(t\)-test. (**A**) Citrine–G\(\alpha\)\({}_{3}\): comparison with the control: **–\(p<0.01\)***-\(p<0.001\), Citrine–G\(\alpha\)\({}_{3}\) mCherry–G\(\beta_{1}\gamma_{2}\) vs. Citrine–G\(\alpha\)\({}_{3}\) mCherry–G\(\beta_{1}\gamma_{2}\) &&&-\(p<0.001\) and mGFP–G\(\alpha\)\({}_{3}\) mCherry–G\(\beta_{1}\gamma_{2}\) vs. mGFP–G\(\alpha\)\({}_{3}\) mCherry–G\(\beta_{1}\gamma_{9}\) ###-\(p<0.001\) (n = 8). (**B**) Citrine–G\(\alpha\)s/s\({}_{\rm IEE}\): comparison with the control: **–\(p<0.01\)***-\(p<0.005\), Citrine–G\(\alpha\)s\({}_{\rm IEE}\)** mCherry–G\(\beta_{1}\gamma_{2}\) vs. Citrine–G\(\alpha\)s\({}_{\rm IEE}\)** mCherry–G\(\beta_{1}\gamma_{9}\)** mCherry–G\(\beta_{1}\gamma_{9}\)** ###-\(p<0.005\) (n = 8). (**C**) In a pull-down assay, G\(\alpha\)s\({}_{\rm IEE}\) was found to interact with G\(\beta_{1}\gamma_{2}\). Recombinant His-tagged mCherry–G\(\beta_{1}\) bound to NiNTA beads were incubated with Citrine–G\(\alpha\)s or Citrine–G\(\alpha\)s\({}_{\rm IEE}\) cell lysates. Beads were precipitated, and the amount of G\(\alpha\) was detected by Western blotting, using an antibody specific to G\(\alpha\)s. The anti-His-tag antibody was used for visualization of His-tagged mCherry–G\(\beta_{1}\). The figure is representative of three independent experiments. Citrine-Gas\({}_{\rm IEK}\) or Citrine-Gas were loaded onto NiNTA agarose baited with His\({}_{6}\)-tagged forms of mCherry-G\(\beta_{1}\) and then the complexes were eluted. Furthermore, purified proteins were detected by immunoblotting with an antibody against the N-terminal epitope of Gas and against the His\({}_{6}\)-tag of G\(\beta_{1}\). Figure 4C summarizes the results of all pull-down experiments, showing the binding of Gas\({}_{\rm IEK}\) to His\({}_{6}\)-tagged G\(\beta_{1}\). No clear signal was observed for the negative cell lysate (Figure 4C), thus confirming that the band detected by the anti-Gas antibody is Gas\({}_{\rm IEK}\) associated with G\(\beta_{1}\) and not endogenous Gas present in the cell lysate. Incubation of the membrane fraction of cells overexpressing Gas\({}_{\rm IEK}\) and G\(\beta_{1Y2}\) with the NiNTA resin resulted in a signal from Gas\({}_{\rm IEK}\), once again indicating the presence of the full G\(\alpha\)\({}_{\rm IEK}\)\(\beta_{1Y2}\) heterotrimer. Since these pull-down experiments are qualitative or at best semi-quantitative in nature, we did not attempt to quantify the relative amounts of the Gas\({}_{\rm IEK}\)\(\beta_{1Y2}\) and Gas\(\beta_{1Y2}\) heterotrimers. Our data strongly suggest that the IEK mutation in Gas is G\(\beta\gamma\) binding-deficient, the heterotrimer is formed less efficiently and has impaired activity but is still able to activate adenylyl cyclase.