# 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
Therefore, an increase in FRET efficiency in our system can be interpreted as a quantitatively higher percentage of complexes formed between a particular G\(\alpha\) and G\(\beta\gamma\), especially when we are comparing trimers with the same G\(\alpha\). Since Citrine in fusion proteins is integrated into other loops in the G\(\alpha\)s and G\(\alpha\)i\({}_{3}\) structure (Figure 3), the structural differences between their heterotrimers should also be considered. Considering the results of intracellular cAMP concentrations obtained for G\(\alpha\)i\({}_{3}\) heterotrimers, it seems reasonable to conclude that FRET efficiency for these complexes may be related to their content at the plasma membrane. The smaller FRET efficiency between Citrine-G\(\alpha\)i\({}_{3}\) and mCherry-G\(\beta\)\({}_{1}\)\(\gamma\)\({}_{9}\) is reflected in a smaller decrease in cAMP concentration when compared to G\(\alpha\)i\({}_{3}\)\(\beta\)\({}_{1}\)\(\gamma\)\({}_{2}\). No differences were found in FRET efficiency between the investigated G\(\alpha\)s heterotrimers, indicating a similar level of G\(\alpha\)s\(\beta\)\({}_{1}\)\(\gamma\)\({}_{2}\) and G\(\alpha\)s\(\beta\)\({}_{1}\)\(\gamma\)\({}_{9}\) complexes at the plasma membrane. However, as shown in Figure 4B, a slight difference in the cAMP concentrations of G\(\alpha\)s\(\beta\)\({}_{1}\)\(\gamma\)\({}_{2}\) and G\(\alpha\)s\(\beta\)\({}_{1}\)\(\gamma\)\({}_{9}\) can be observed, although it is not statistically significant and is smaller than between G\(\alpha\)i\({}_{3}\) heterotrimers. Thus, even if there is a difference in the concentrations of these complexes at the plasma membrane, it is not substantial. On the contrary, there are significant differences in FRET signals between G\(\alpha\)s and G\(\alpha\)i\({}_{3}\) heterotrimers which cannot be uniquely attributed to only a single factor. Most probably the FRET signal reflects a mixture of effects, i.e., a potentially lower percentage of trimers composed of G\(\alpha\)s and G\(\beta\)\({}_{1}\)\(\gamma\)\({}_{2}\) or G\(\beta\)\({}_{1}\)\(\gamma\)\({}_{9}\) dimers than G\(\alpha\)i\({}_{3}\) heterotrimers, and structural differences between these heterotrimers due to different localizations of fluorescence donors (Figure 3). The estimated difference in the distance separating mCherry-G\(\beta\)\({}_{1}\) and Citrine-G\(\alpha\)s is, on average, 6.5 A longer than in the G\(\alpha\)i\({}_{3}\) heterotrimers. Thus, the energy transfer efficiency in the G\(\alpha\)s heterotrimers should be lower by about 7% than in G\(\alpha\)i\({}_{3}\) heterotrimers, assuming Forster distance R\({}_{0}\) of 56.6 A for this pair of fluorophores [38; 39]. Figure 2: FLIM–FRET of G\(\alpha\)s\(\beta\)\({}_{1}\)\(\gamma\)\({}_{2}\), G\(\alpha\)s\(\beta\)\({}_{1}\)\(\gamma\)\({}_{9}\), G\(\alpha\)i\({}_{3}\)\(\beta\)\({}_{1}\)\(\gamma\)\({}_{2}\), and G\(\alpha\)i\({}_{3}\)\(\beta\)\({}_{1}\)\(\gamma\)\({}_{9}\) heterotrimers. (**A**) HEK 293 cells were transiently transfected with Citrine–G\(\alpha\) alone or both mCherry–G\(\beta\)\(\gamma\) and Citrine–G\(\alpha\). Fluorescence lifetimes are presented in a continuous pseudo-color scale representing time values ranging from 2.7 (blue) to 3.6 (red) or 3.7 ns (red). (**B**) Box-and-whisker plots of the fluorescence lifetime \(\tau_{1}\) of energy donor (Citrine–G\(\alpha\)) and donor in the presence of acceptor (mCherry–G\(\beta\)\(\gamma\)). The median is shown as a line in the box, while the bottom and top boundaries represent the lower and upper quartile, respectively. Statistical significance of the difference in the donor fluorescence lifetimes \(\tau_{1}\) detected in the absence and presence of energy acceptor using Mann–Whitney \(U\) test (* \(p<0.02\), ** \(p<0.002\), *** \(p<0.0002\)).