The polybasic region in Gαi proteins: Relevant or not? Insights from Gαi3 research.

Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several signals, including modification by fatty acid moieties, interaction with the Gßγ complex, and, as observed in some Gα proteins, the presence of basic amino acid residues in the N-terminal region. In this work, we focused on investigating the influence of the polybasic region on the localization and function of a representative member of the Gαi family, Gαi3. Through the use of confocal microscopy and fluorescence lifetime microscopy, we showed that, in the case of this protein, neutralizing the positive charge does not significantly affect its abundance in the cell membrane. However, it does affect its spatial arrangement concerning the dopamine D2 receptor and influences inhibitory effect of Gαi3 on intracellular cAMP production triggered by D2 receptor stimulation. Moreover, in this work, we have shown, for the first time, that nonlipidated Gαi3 binds to negatively charged lipids through electrostatic interactions, and membrane fluidity plays a significant role in this interaction.

Beyond the G protein α subunit: investigating the functional impact of other components of the Gαi3 heterotrimers.

BACKGROUND: Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gß and Gγ subunits and the effects of different Gßγ dimer compositions on GPCR signaling are poorly understood. In this study, we aimed to investigate the subcellular localization and functional response of Gαi3-based heterotrimers with different combinations of Gß and Gγ subunits. METHODS: Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi3 in combination with Gß1 or Gß2 heterotrimers, along with representative Gγ subunits. Furthermore, fluorescence lifetime imaging microscopy (FLIM-FRET) was used to investigate the nanoscale distribution of Gαi3-based heterotrimers in the plasma membrane, specifically with the dopamine D2 receptor (D2R). In addition, the functional response of the system was assessed by monitoring intracellular cAMP levels and conducting bioinformatics analysis to further characterize the heterotrimer complexes. RESULTS: Our results show that Gαi3 heterotrimers mainly localize to the plasma membrane, although the degree of colocalization is influenced by the accompanying Gß and Gγ subunits. Heterotrimers containing Gß2 showed slightly lower membrane localization compared to those containing Gß1, but certain combinations, such as Gαi3ß2γ8 and Gαi3ß2γ10, deviated from this trend. Examination of the spatial arrangement of Gαi3 in relation to D2R and of changes in intracellular cAMP level showed that the strongest functional response is observed for those trimers for which the distance between the receptor and the Gα subunit is smallest, i.e. complexes containing Gß1 and Gγ8 or Gγ10 subunit. Deprivation of Gαi3 lipid modifications resulted in a significant decrease in the amount of protein present in the cell membrane, but did not always affect intracellular cAMP levels. CONCLUSION: Our studies show that the composition of G protein heterotrimers has a significant impact on the strength and specificity of GPCR-mediated signaling. Different heterotrimers may exhibit different conformations, which further affects the interactions of heterotrimers and GPCRs, as well as their interactions with membrane lipids. This study contributes to the understanding of the complex signaling mechanisms underlying GPCR-G-protein interactions and highlights the importance of the diversity of Gß and Gγ subunits in G-protein signaling pathways. Video Abstract.

The Gαi protein subclass selectivity to the dopamine D2 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 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. Video abstract.

'Shotgun' proteomic analyses without alkylation of cysteine.

It is a common belief that reduction of disulfide bridges and alkylation of thiols in proteins are indispensable steps in proteomic sample preparation. Since this chemical procedure is often incomplete and prone to side reactions we reexamined its importance. We found that reduction and alkylation do not increase the depth of analysis and quality of proteomic quantification and therefore these steps are not essential in 'shotgun'-type investigations of proteomes. Moreover, we found that compared to a standard procedure using iodoacetamide for thiol-alkylation, sample preparation under conditions protecting thiols from oxidation improves quality of peptides and allows identifying of 10-20% more peptides and proteins. Excluding thiol-alkylation from proteomic sample preparation shortens the workflows and decreases the probability of biases resulting from occurrence of artificially modified peptides.

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting.

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various heterotrimeric complexes. In a previous study, we found that Gαs and Gαi3 prefer different types of membrane-anchor and subclass-specific lipid domains. In the present report, we examine the role of distinct Gγ subunits in the membrane localization and spatiotemporal dynamics of Gαs and Gαi3 heterotrimers. We characterized lateral diffusion and G-protein subunit interactions in living cells using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM), respectively. The interaction of Gγ subunits with specific lipids was confirmed, and thus the modulation of heterotrimeric G-protein localization. However, the Gα subunit also modulates trimer localization, and so the membrane distribution of heterotrimeric G-proteins is not dependent on Gγ only.