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

Study of D1 dopamine receptor interaction with G protein alpha subunit

Receptory sprzężone z białkami G stanową największą rodzinę białek błonowych. W ludzkim genomie zidentyfikowano ponad 800 genów kodujących te receptory. Receptory metabotropowe aktywowane są przez zewnętrzny sygnał w formie ligandu lub innego mediatora. Związanie ligandu wywołuje zmiany konformacyjne receptora, skutkujące aktywacją białka G. Mechanizmy molekularne oddziaływania receptorów z białkami G pozostają nierozpoznane. Wciąż istnieją pytania bez odpowiedzi: czy białko G wiąże się z receptorem przed czy po aktywacji samego receptora, czy oligomeryzacja receptorów metabotropowych wywołuje zmiany w rozpoznawaniu liganda czy w związaniu białka G.Celem niniejszej pracy była weryfikacja hipotezy dotyczącej prekompleksowania receptorów i białek G. Odziaływanie badano w układzie modelowym złożonym z receptora dopaminowego D1 oraz podjednostki αsL białka G. W badaniach wykorzystano barwnik FlAsH rozpoznający motyw sekwencji wprowadzony do trzeciej pętli wewnątrzkomórkowej (IC-3) oraz C-końca receptora dopaminowego. Metoda znakowania barwnikiem FlAsH polega na wiązaniu się małej, arsenowej pochodnej fluoresceiny do sześcioaminokwasowej sekwencji Cys-Cys-Pro-Gly-Cys-Cys (CCPGCC). Uzyskany stosunek sygnału do szumu dla znacznika FlAsH jest bardzo niski. W celu potwierdzenia specyficznego znakowania potrzebne są dalsze optymalizacje rozpoznawanej sekwencji i/lub warunków, w których przeprowadza się znakowanie. Tworzenie kompleksów receptora z białkiem G badano poprzez pomiary rezonansowego transferu energii techniką obrazowania czasów życia fluorescencji (FRET-FLIM). W celu detekcji prekompleksów wykorzystano badane białka połączone z białkami fluorescencyjnymi Citrine oraz mCherry. Uzyskane wyniki potwierdziły oddziaływanie pomiędzy białkami w badanym układzie. Podjęto także próbę stworzenia układu modelowego na bazie fluorescencyjnego dopełnienia (BiFC) do badania homo- i heterodimeryzacji receptorów dopaminowych D2 i/lub D1. W celu ułatwienia reasocjacji fluoroforu wydłużono C-końcowy fragment receptora D2. Układ ten pozwolił na obserwację homodimerów receptora D2 w komórkach HEK293.G protein-coupled receptors represent largest family of integral membrane proteins. In the human genome were identified more than 800 GPCR genes. The G protein-coupled receptor is activated by an external signal in the form of a ligand or other signal mediator. This creates a conformational change in the receptor, causing activation of a G protein. Molecular mechanism of receptor interactions with G proteins remains unrecognized. There are still unanswered questions: whether G proteins couple before or after receptor activation, whether GPCR oligomerization involves changes in ligand recognition or G protein-coupling. This thesis summarizes an attempt to test the hypothesis of receptor-G protein precoupling using D1 dopamine receptor and αsL subunit of G protein. FlAsH-EDT2 was used as a site-specific label of the III cytoplasmic loop and C-terminal of the dopamine receptor. This method is based on the binding of small fluorescein derivative, called fluorescein arsenical hairpin binder (FlAsH), to a short peptide sequence Cys-Cys-Pro-Gly-Cys-Cys (CCPGCC). The signal to noise ratio of the FlAsH dye is relatively low. Further optimization of labeling motif and/or labeling conditions appears to be required if the specificity of labeling is to be improved. Precoupling was investigated by Forster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM). Chimeras with the fluorescent proteins Citrine and mCherry were used to detect precoupling. The results confirmed interaction between proteins.An attempt was also made to create a fluorescence complementation-based system for the study of D2 and/or D1 dopamine receptors homo- and heterodimers. The C-terminus of D2 dopamine receptor was extended to facilitate fluorescence complementation. D2 homodimers have been visualized in HEK293 cells

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

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex

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

Abstract 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 Gβ and Gγ subunits and the effects of different Gβγ 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 Gβ and Gγ subunits. Methods Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi3 in combination with Gβ1 or Gβ2 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 Gβ and Gγ subunits. Heterotrimers containing Gβ2 showed slightly lower membrane localization compared to those containing Gβ1, but certain combinations, such as Gαi3β2γ8 and Gαi3β2γ10, 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 Gβ1 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 Gβ and Gγ subunits in G-protein signaling pathways. Video Abstrac

New insights into the model of dopamine D_{1} receptor and G-proteins interactions

AbstractThe 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 9–10nm