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

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 Gαi 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 D long receptor isoform (DR) toward the Gαi, Gαi, and Gαi 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 Gαi subunit and the singular distribution of the Gαi subunit in the plasma membrane. The cell membrane partitioning of distinct Gαi heterotrimers with dopamine receptor correlated very well with the efficiency of DR-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. Video abstract.