# G\(\gamma\) and G\(\alpha\) Identity Dictate a G-Protein Heterotrim Plasma Membrane Targeting
## 3 Results  
### Functionality of Fluorescently-Tagged G-Proteins  
We have shown previously that stimulatory G\(\alpha\)s and inhibitory G\(\alpha\)i\({}_{3}\) subunits are located in distinct types of the membrane domains, depending on their specific activation state [22; 25]. In previous experiments we reported that co-transfection of G\(\alpha\)s or G\(\alpha\)i\({}_{3}\) with the G\(\beta\)i\({}_{1}\)Y\({}_{2}\) dimer and the dopamine D\({}_{1}\) receptor influences the membrane location of G\(\alpha\)s and, to a lesser extent, of the G\(\alpha\)i\({}_{3}\)[25]. In their presence, G\(\alpha\), complexed with G\(\beta\)i\({}_{1}\)Y\({}_{2}\), relocates outside the liquid-ordered membrane domains. Because of the possibility that this result arose from the concurrent actions of the G\(\beta\)i\({}_{1}\)Y\({}_{2}\) dimer and the D\({}_{1}\) receptor, in the present study we focused solely on the effect of G\(\beta\)Y. More specifically, we addressed the question whether the localization of the heterotrim at the plasma membrane was controlled only by the G\(\beta\)Y dimer. Tagging proteins of interest with FP might reduce their biological function due to undesired conformational changes or steric hindrance introduced by the tags. In order to avoid this, we selected loop-tagged G\(\alpha\)s and G\(\alpha\)i\({}_{3}\) and amino-terminal-tagged G\(\beta\)i\({}_{1}\) for further optimization and analyses. The FP sequence was inserted into the L1 loop in the G\(\alpha\)s subunit (the loop from Glu\({}_{71}\) to Ser\({}_{82}\) residue (G.h1 ha.7-G.h1 ha.18) was exchanged), and into the second loop (\(\alpha\)BC loop) within the helical domain of the G\(\alpha\)i\({}_{3}\) subunit (FP sequence cloned after Ala\({}_{114}\) residue (H.hbhc.2)). All investigated proteins displayed plasma membrane localization as shown by the fluorescence images, confirming that the presence of tags does not disturb their membrane-binding ability (Figure 1). The loop fusion G\(\alpha\)s has been reported to be functional [27]. Additionally, intracellular cAMP level was measured in order to check the functional activity of all designed fusion proteins. The G\(\alpha\)s and G\(\alpha\)i\({}_{3}\) subunits are distinct and provide the specificity for activation and inhibition, respectively, of adenylyl cyclase. Depending on the G\(\alpha\) subclass, HEK 293 cells were co-transfected with adenosine A\({}_{2A}\) or the dopamine D\({}_{2}\) receptor. In response to extracellular stimuli with a suitable agonist (2-phenylaminoadenosine or sumanirole), a rapid increase or reduction in the production of cAMP was detected for G\(\alpha\)s or G\(\alpha\)i\({}_{3}\), respectively (Figure 1A,B). These results indicate that the tagged G-protein heterotrimers are signaling-active. The G\(\alpha\)s heterotrimers were capable of more than eight-fold induction in cAMP over the basal level. In turn, G\(\alpha\)i\({}_{3}\) heterotrimers showed pronounced inhibitory effect. G\(\alpha\)i\({}_{3}\), tagged with Citrine or mGFP, was examined and no significant differences in cAMP production between these fusion proteins were found. Intriguingly, trimers composed of G\(\beta\)i\({}_{1}\)Y\({}_{9}\) changed the basal cAMP level in HEK 293 cells less efficiently than trimers consisting of G\(\beta\)i\({}_{1}\)Y\({}_{2}\), especially G\(\alpha\)i\({}_{3}\)\(\beta\)i\({}_{1}\)Y\({}_{9}\). This finding may be explained by a potentially less effective formation of the full heterotrimer G\(\alpha\)\(\beta\)i\({}_{1}\)Y\({}_{9}\) complex than G\(\alpha\)g\({}_{1}\)Y\({}_{2}\). It was previously reported that some combinations of G\(\alpha\) and G\(\beta\)Y were less stable and dissociated over longer periods of time [28]. On the other hand, the coupling of the G\(\alpha\)\(\beta\)i\({}_{1}\)Y\({}_{2}\) protein to dopamine D\({}_{2}\)R or adenosine A\({}_{2A}\)R, in response to agonist activation, may be more productive than interaction with G\(\alpha\)g\({}_{1}\)Y\({}_{9}\).