The Gαi protein subclass selectivity to the dopamine D2 receptor is also decided by their location at the cell membrane

Abstract

Background G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins plays an important role in the cellular regulation of responses to external stimuli. Despite intensive structural research, the mechanism underlying the receptor–G protein coupling of closely related subtypes of Gαi remains unclear. In addition to the structural changes of interacting proteins, the interactions between lipids and proteins seem to be crucial in GPCR-dependent cell signaling due to their functional organization in specific membrane domains. In previous works, we found that Gαs and Gαi3 subunits prefer distinct types of membrane-anchor lipid domains that also modulate the G protein trimer localization. In the present study, we investigated the functional selectivity of dopamine D2 long receptor isoform (D2R) toward the Gαi1, Gαi2, and Gαi3 subunits, and analyzed whether the organization of Gαi heterotrimers at the plasma membrane affects the signal transduction. Methods We characterized the lateral diffusion and the receptor–G protein spatial distribution in living cells using two assays: fluorescence recovery after photobleaching microscopy and fluorescence resonance energy transfer detected by fluorescence-lifetime imaging microscopy. Depending on distribution of data differences between Gα subunits were investigated using parametric approach–unpaired T-test or nonparametric–Mann–Whitney U test. Results Despite the similarities between the examined subunits, the experiments conducted in the study revealed a significantly faster lateral diffusion of the Gαi2 subunit and the singular distribution of the Gαi1 subunit in the plasma membrane. The cell membrane partitioning of distinct Gαi heterotrimers with dopamine receptor correlated very well with the efficiency of D2R-mediated inhibition the formation of cAMP. Conclusions This study showed that even closely related subunits of Gαi differ in their membrane-trafficking properties that impact on their signaling. The interactions between lipids and proteins seem to be crucial in GPCR-dependent cell signaling due to their functional organization in specific membrane domains, and should therefore be taken into account as one of the selectivity determinants of G protein coupling.

Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

“…In addition to lipid modifications of the Gα subunits, a polybasic motif at the Nterminal helical region has been shown to influence their membrane binding and localization as well as interaction with the Gβγ dimer [30,37,90,91]. Polybasic motifs are present in a multitude of plasma membrane-targeted proteins, including domains specialized in interacting with cell membranes [92][93][94][95].…”

“…Interestingly, a recent report addressing membrane localization and distribution of the Gαi family members, namely Gαi 1 , Gαi 2 , and Gαi 3, showed that the N-terminal polybasic cluster affects its interaction with the plasma membrane [30]. Nevertheless, the electrostatic potential maps of Gαi subunits are similar and contain negative patches that produce the Gαt membrane repulsion [38].…”

“…This distortion is manifested as a high level of local water penetration inside the membrane and can lead to a decrease in the thickness of the bilayer as well as affect long-range interactions [102]. The reduced membrane mobility of Gαi 1 corresponds to the presence of a larger number of arginine residues in the polybasic motif compared to that in the other two Gαi subunits [30].…”

“…The specific interaction of Gαq with caveolin allowed researchers to conclude that caveolin cavities are the major region of residence of this subunit [157]. In our research, we focused on the localization and protein interactions of the dopamine signal transduction pathway: D 1 and D 2 receptors and cognate G proteins, Gαs and Gαi 1-3 subunits [30,31,135,158]. To study the mutual distribution of the receptor-G protein complex, we used microscopy techniques to measure the distance between the proteins and their lateral diffusion.…”

“…This knowledge is very important as it shows how different signaling proteins can be precisely targeted at a predestined location by incorporating well-defined modifications. We focused on the Gα subunits as new data revealed considerable diversity in membrane trafficking properties of closely related Gα subunits [29,30]. Furthermore, it has been found that the membrane distribution of heterotrimeric G proteins is not dependent only on Gβγ, as was commonly believed.…”