Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Kraków, Poland.
Cell Commun Signal. 2020 Dec 11;18(1):189. doi: 10.1186/s12964-020-00685-9.
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 Gαi 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 D long receptor isoform (DR) toward the Gαi, Gαi, and Gαi subunits, and analyzed whether the organization of Gαi heterotrimers at the plasma membrane affects the signal transduction.
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.
Despite the similarities between the examined subunits, the experiments conducted in the study revealed a significantly faster lateral diffusion of the Gαi subunit and the singular distribution of the Gαi subunit in the plasma membrane. The cell membrane partitioning of distinct Gαi heterotrimers with dopamine receptor correlated very well with the efficiency of DR-mediated inhibition the formation of cAMP.
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.
通过异三聚体 G 蛋白的 G 蛋白偶联受体 (GPCR) 信号转导在细胞对外界刺激反应的调节中起着重要作用。尽管进行了深入的结构研究，但对于密切相关的 Gαi 亚型的受体-G 蛋白偶联机制仍不清楚。除了相互作用蛋白的结构变化外，由于脂质和蛋白质在特定膜域中的功能组织，它们之间的相互作用似乎在 GPCR 依赖性细胞信号转导中至关重要。在以前的工作中，我们发现 Gαs 和 Gαi 亚基优先选择不同类型的膜锚定脂质域，这些脂质域也调节 G 蛋白三聚体定位。在本研究中，我们研究了多巴胺 D 长受体同工型 (DR) 对 Gαi、Gαi 和 Gαi 亚基的功能选择性，并分析了质膜上 Gαi 异三聚体的组织是否影响信号转导。
我们使用两种测定法：光漂白后荧光恢复显微镜和荧光寿命成像显微镜检测的荧光共振能量转移，来表征活细胞中的侧向扩散和受体-G 蛋白空间分布。根据数据分布的差异，使用参数方法（未配对 T 检验）或非参数方法（Mann-Whitney U 检验）来研究 Gα 亚基之间的差异。
尽管被检查的亚基具有相似性，但研究中进行的实验表明 Gαi 亚基的侧向扩散速度明显更快，并且 Gαi 亚基在质膜中呈单一分布。不同 Gαi 异三聚体与多巴胺受体的细胞膜分配与 DR 介导的 cAMP 形成抑制效率密切相关。
本研究表明，即使是密切相关的 Gαi 亚基在其膜运输特性上也存在差异，这会影响其信号转导。由于脂质和蛋白质之间的相互作用在特定膜域中的功能组织，它们在 GPCR 依赖性细胞信号转导中似乎至关重要，因此应将其作为 G 蛋白偶联的选择性决定因素之一加以考虑。
