背景:G蛋白偶联受体(GPCRs)和G蛋白之间的特异性相互作用在介导信号传导事件中起关键作用。虽然对Gα亚基的受体偏好几乎没有疑问,对特定Gβ和Gγ亚基的偏好以及不同Gβγ二聚体组成对GPCR信号传导的影响了解甚少。在这项研究中,我们旨在研究具有不同Gβ和Gγ亚基组合的基于Gαi3的异源三聚体的亚细胞定位和功能反应。
方法:使用活细胞成像显微镜和共定位分析来研究Gαi3与Gβ1或Gβ2异源三聚体的亚细胞定位,以及代表性的Gγ亚基。此外,荧光寿命成像显微镜(FLIM-FRET)用于研究基于Gαi3的异源三聚体在质膜中的纳米级分布,特别是多巴胺D2受体(D2R)。此外,通过监测细胞内cAMP水平并进行生物信息学分析以进一步表征异源三聚体复合物来评估系统的功能反应。
结果:我们的结果表明,Gαi3异源三聚体主要定位于质膜,尽管共定位的程度受伴随的Gβ和Gγ亚基的影响。与含有Gβ1的那些相比,含有Gβ2的异三聚体显示出较低的膜定位,但某些组合,如Gαi3β2γ8和Gαi3β2γ10,偏离了这一趋势。检查Gαi3相对于D2R的空间排列和细胞内cAMP水平的变化表明,对于受体与Gα亚基之间距离最小的三聚体,观察到最强的功能反应,即含有Gβ1和Gγ8或Gγ10亚基的复合物。剥夺Gαi3脂质修饰导致细胞膜中存在的蛋白质数量显着减少,但并不总是影响细胞内cAMP水平。
结论:我们的研究表明,G蛋白异源三聚体的组成对GPCR介导的信号传导的强度和特异性具有显着影响。不同的异源三聚体可能表现出不同的构象,进一步影响异源三聚体和GPCRs的相互作用,以及它们与膜脂的相互作用。这项研究有助于理解GPCR-G蛋白相互作用的复杂信号机制,并强调Gβ和Gγ亚基在G蛋白信号通路中的多样性的重要性。视频摘要。
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.
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.
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.
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 Abstract.