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 the interaction between G protein alpha i3 subunit and long isoform of dopamine D2 receptor using fluorescence spectroscopy

Receptory sprzężone z białkami G (GPCR) są jedną z najbardziej rozpowszechnionych grup receptorów błonowych w komórkach. Są związane z odpowiedzią komórki na szereg różnorodnych sygnałów w tym neurotransmiterów. Receptory dopaminowe należą do klasy A receptorów sprzężonych z białkami G, a zaburzenia ich funkcjonowania prowadzą do takich zaburzeń jak schizofrenia, choroba Parkinsona czy depresja. Receptory te oddziałują z trimerycznymi białkami G, w przypadku receptora dopaminowego D2 z klasą Gαi. Pierwsze etapy odpowiedzi na aktywację receptora D2 związane z oddziaływaniem z białkiem G, w szczególności z ich wzajemną lokalizacją w błonie komórkowej, nie zostały do tej pory dokładnie scharakteryzowane. Celem pracy było zbadanie czy w błonie komórkowej izoforma długa receptora dopaminowego D2 i podjednostka αi3 białka G wykazują podobną lokalizację. Badania prowadzono poprzez obserwację zmian rezonansowego transferu energii metodą obrazowania czasów życia fluorescencji (FLIM-FRET) w warunkach podstawowych oraz po stymulacji receptora pełnymi agonistami: rotigotiną lub bromokryptyną.G protein coupled receptors (GPCR) represent one of the largest group of membrane receptors. They’re connected with variety of stimuli within cell including neurotransmitters. Dopamine receptors represents class A of GPCR and are crucial in the pathogenesis diseases such as schizophrenia, Parkinson disease or depression. Those receptors interact with trimeric G proteins, in case of dopamine D2 receptors interaction occurs within Gαi class of G proteins. The localisation of the receptor and G proteins within cell membrane and the initial events after activation of D2 receptor has not yet been characterised in details. The purpose of this work was to investigate if there occurs colocalization of the D2 receptor (long isoform) and αi3 subunit of G protein in the cell membrane. The research was carried out by examination of changes in Forster resonance energy transfer (FRET). The measurements were performed using fluorescence lifetime imaging microscopy (FLIM) in basal conditions and after stimulation of the receptor by full agonists: rotigotine or bromocriptine

Effect of low temperature on the level of UCP-1 in brown adipose tissue in the CMKRL1 and wild type mice

Najważniejszą funkcją brązowej tkanki tłuszczowej jest udział w termogeneziebezdrżeniowej. Odbywa się ona dzięki dysypacji energii, na skutek rozprzęgana łańcuchaoddechowego w mitochondriach. Jednym z najważniejszych białek związanych z tym procesem jesttermogenina (UCP1). Chemeryna jest białkiem o ugruntowanej roli w procesachimmunologicznych, ponadto jest czynnikiem wydzielanym przez adipocyty oraz wpływa na ichfunkcjonowanie. Celem pracy jest zbadanie wpływu braku receptora dla chemeryny na ekspresjęUCP1 w brązowej tkance tłuszczowej, który może mieć znaczenie w regulacji termogenezyu ssaków.The most important function of brown adipose tissue in organisms is the nonshiveringthermogenesis. Process occurs owing to dysypation of energy in mitochondria. One of the mostimportant protein involved in that process is termogenin (UCP1 – uncoupling protein 1). Chemerinis a protein involved in immunological processes, moreover is an adipokine which influenceon functioning of the adipose tissue. The aim of this work is study of impact of lack the chemerinreceptor on expression of UCP1 in brown adipose tissue, which may be important in regulationof mammals thermogenesis

G\gamma and G\alpha 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

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