G Protein-Coupled Receptor Dimerization-What Next?

Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.

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.

Bradykinin B2 and dopamine D2 receptors form a functional dimer.

In recent years a wide range of studies have shown that G protein-coupled receptors modulate a variety of cell functions through the formation of dimers. For instance, there is growing evidence for the dimerization of bradykinin or dopamine receptors, both as homodimers and heterodimers. A discovery of direct interactions of angiotensin II receptors with bradykinin 2 receptor (B2R) or dopamine D2 (D2R) receptor has led to a hypothesis on a potential dimerization between two latter receptors. In this study, we have demonstrated a constitutive colocalization of receptors on the membranes of HEK293 cells transiently transfected with plasmid vectors encoding B2R and D2R, fused with fluorescent proteins. The receptor colocalization was significantly enhanced by specific agonists of B2R or D2R after 5min following the addition, whereas simultaneous stimulation with these agonists did not influence the B2R/D2R colocalization level. In addition, B2R-D2R heterodimerization was confirmed with FLIM-FRET technique. The most characteristic signaling pathways for B2R and D2R, dependent on intracellular Ca2+ and cAMP concentration, respectively, were analyzed in cells presenting similar endogenous expression of B2R and D2R. Significant changes in receptors' signaling were observed after simultaneous stimulation with agonists, suggesting transformations in proteins' conformation after dimerization. The evidence of B2R-D2R dimerization may open new perspectives in the modulation of diverse cellular functions which depend on their activation.

The role of cholesterol and sphingolipids in the dopamine D1 receptor and G protein distribution in the plasma membrane.

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein-lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi3 prefer different types of membrane domains they are both co-localized with the D1 receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi3 and their heterotrimers, as well as the D1 receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-ß-cyclodextrin or Fumonisin B1 led to the disruption of cholesterol-sphingolipids containing domains and changed the diffusion of Gαi3 and the D1 receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi3 prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D1 receptor exists in several different membrane localizations, depending on the receptor's conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.

Intra- and intersubunit changes accompanying thermal activation of the HtrA2(Omi) protease homotrimer.

HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limits access both to the PDZ peptide (carboxylate) binding site and to the PD catalytic center. The molecular mechanism of activation is not well understood. To further the knowledge of HtrA2 thermal activation we monitored the dynamics of the PDZ-PD interactions during temperature increase using tryptophan-induced quenching (TrIQ) method. The TrIQ results suggested that during activation the PDZ domain changed its position versus PD inside a subunit, including a prominent change affecting the L3 regulatory loop of PD, and also changed its interactions with the PD of the adjacent subunit (PD*), specifically with its L1* regulatory loop containing the active site serine. The α5 helix of PDZ was involved in both, the intra- and intersubunit changes of interactions and thus seems to play an important role in HtrA2 activation. The amino acid substitutions designed to decrease the PDZ interactions with the PD or PD* promoted protease activity at a wide range of temperatures, which supports the conclusions based on the TrIQ analysis. The model presented in this work describes PDZ movement in relation to PD and PD*, resulting in an increased access to the peptide binding and active sites, and conformational changes of the L3 and L1* loops.

New insights into the model of dopamine D1 receptor and G-proteins interactions.

The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane. Using fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) or fluorescence recovery after photobleaching (FRAP) microscopy, we did not detect constitutive preassociated complex between D1 receptor and G-protein in the absence of receptor activation. Our work suggests that D1 receptor alters the distribution of Gαs and Gαi3 subunits inside the membrane. We also find that non-activated D1 receptor and Gαs or Gαi3 are present in the cell membrane within the same membrane microdomains in the proximity of about 9-10 nm.

Comparative EPR studies on lipid bilayer properties in nanodiscs and liposomes.

Studies of the membrane proteins suggest their close interaction with the lipid surroundings. Membrane proteins and their activities are affected by the composition and structure of the lipid bilayer. therefore adequate surroundings for studied protein are crucial for the model membrane to ensure its biological relevance. In recent years nanodiscs which are small fragments of lipid bilayer stabilised by derivatives of apolipoprotein, called membrane scaffold protein ( MSP), have been established as alternative tool in structural and functional studies of membrane proteins. In this study, the influence MSP of different length on structure and dynamics of DMPC and POPC bilayer was investigated and compared to bilayer present in liposomes. EPR spectroscopy technique using different PC-based spin probes was employed to show cholesterol-like organising effect of MSPs on lipid bilayer, thus giving a better insight into the nanodiscs model membrane structure, and its possible implications in the research of membrane protein applications.

The LA loop as an important regulatory element of the HtrA (DegP) protease from Escherichia coli: structural and functional studies.

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA' (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.

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.