# G\(\gamma\) and G\(\alpha\) Identity Dictate a G-Protein Heterotrim Plasma Membrane Targeting
## 4 Discussion  
In this study, we have combined two live-cell imaging microscopic methods, FLIM-FRET and FRAP, to investigate the molecular-level assembly properties and the trafficking dynamics of G-protein heterotrimers within the cell membrane. The membrane dynamics of heterotrimer complexes were monitored by FRAP. The studied G\(\alpha\)s and G\(\alpha\)i\({}_{3}\) subunits are expressed in most types of cells. Similarly, the G\(\beta_{1}\) and G\(\gamma_{2}\) subunit expression is ubiquitous, whereas G\(\gamma_{9}\) is mostly present in olfactory epithelium [1]. Although much progress has been made in understanding the molecular details of how G-proteins interact with GPCRs and regulate the activity of their downstream targets, it is less clear how activated GPCRs initiate this process and what the trafficking pathway of the heterotrimeric G-proteins is within the plasma membrane. The role of the plasma membrane lateral organization in the spatiotemporal distribution of GPCRs and G-proteins appears to be essential in the process of extracellular signal transduction. However, the molecular basis for the interaction of signaling molecules with lipids is still not fully understood but seems to be of key importance in understanding the functional selectivity and activation speed of cellular responses to G-protein activation. Previous evidence supports the direct role of the G\(\gamma\) subunit in G-protein activation by a receptor [41; 42; 43], and also suggests G\(\gamma\) diversity as a crucial modulator of G-protein membrane localization behaviors as well as trimer assembly [44; 45; 46]. Using the same FLIM-FRET and FRAP approach, we previously reported that preferences in localization within the membrane of the stimulatory and inhibitory G\(\alpha\) subunits, G\(\alpha\)s and G\(\alpha\)i\({}_{3}\), are different and further modulated by the dopamine D\({}_{1}\) receptor, as well as by the G\(\beta_{1}\gamma_{2}\) dimer [22; 25]. The association of G\(\beta_{1}\gamma_{2}\) with the GDP-bound G\(\alpha\) subunit translocates G-proteins outside the liquid-ordered membrane domains, which is particularly evident for G\(\alpha\)s [25]. Here we confirmed the ability of the G\(\gamma\) subunit to bind specific lipids, and consequently, to influence the membrane localization of the full heterotrimeric complex of the G-protein; but our results further showed that its membrane distribution was not only G\(\gamma\)-subclass dependent. In this study, we examined the impact of two distinct G\(\gamma\) subunits, G\(\gamma_{2}\), and G\(\gamma_{9}\), on the membrane localization of G-proteins. These G\(\gamma\) differ, among others, in membrane anchors at carboxyl-terminal cysteine in the CaaX motif. The prenyl group promotes tethering of G\(\beta\gamma\) complexes to membranes, sorting of particular lipid domains, as well as playing a role in the translocation properties of the G\(\beta\gamma\) dimer and effector activation [47; 48]. The G\(\gamma_{2}\) belongs to the group of slowest translocating G\(\gamma\) subunits (t\({}_{1/2}\)\(\sim\)130 s) whereas G\(\gamma_{9}\) belongs to the fastest one (t\({}_{1/2}\)\(\sim\) 10 s) [19; 20]. The prenylation of G\(\gamma_{2}\) with a 20-carbon geranylgeranyl lipid, together with positively charged residues in the C-terminal domain, provides it with a higher affinity for the plasma membrane than G\(\gamma_{9}\) with the 15-carbon farnesyl lipid attachment and fewer positively charged residues. The presence of a five-residue or six-residue cluster of positively charged amino acids in the pre-CaaX region modulates G\(\gamma\)-membrane interactions, strengthening the plasma membrane affinity [20], and also governs the membrane-interacting ability of G\(\beta\gamma\)[42]. Considering how many possible G\(\beta\gamma\) and G\(\alpha\) combinations exist, a key question in G-protein signaling is whether the plasma membrane location of heterotrimers composed of distinct subunits is only G\(\beta\gamma\)-dependent.