Nishimura Akiyuki, Nishiyama Kazuhiro, Ito Tomoya, Mi Xinya, Kato Yuri, Inoue Asuka, Aoki Junken, Nishida Motohiro
Division of Cardiocirculatory Signaling, National Institute for Physiological Sciences (NIPS), National Institutes of Natural Sciences, Okazaki 444-8787, Japan.
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki 444-8787, Japan.
Cells. 2025 Feb 3;14(3):216. doi: 10.3390/cells14030216.
G protein-coupled receptors (GPCRs) exist in the conformational equilibrium between inactive state and active state, where the proportion of active state in the absence of a ligand determines the basal activity of GPCRs. Although many GPCRs have different basal activity, it is still unclear whether physiological stresses such as substrate stiffness affect the basal activity of GPCRs. In this study, we identified that purinergic P2Y receptor (P2YR) induced spontaneous Ca oscillation without a nucleotide ligand when cells were cultured in a silicon chamber. This P2YR-dependent Ca oscillation was absent in cells cultured in glass dishes. Coating substrates, including collagen, laminin, and fibronectin, did not affect the P2YR spontaneous activity. Mutation of the extracellular Arg-Gly-Asp (RGD) motif of P2YR inhibited spontaneous activity. Additionally, extracellular Ca was required for P2YR-dependent spontaneous Ca oscillation. The GPCR screening assay identified cells expressing 10 GPCRs, including purinergic P2YR, P2YR, and P2YR, that exhibited spontaneous Ca oscillation under cell culture soft substrate. Our results suggest that stiffness of the cell adhesion surface modulates spontaneous activities of several GPCRs, including P2YR, through a ligand-independent mechanism.
G蛋白偶联受体（GPCRs）存在于非活性状态和活性状态之间的构象平衡中，在没有配体的情况下活性状态的比例决定了GPCRs的基础活性。尽管许多GPCRs具有不同的基础活性，但诸如底物硬度等生理应激是否会影响GPCRs的基础活性仍不清楚。在本研究中，我们发现当细胞在硅室中培养时，嘌呤能P2Y受体（P2YR）在没有核苷酸配体的情况下诱导自发钙振荡。在玻璃培养皿中培养的细胞中不存在这种依赖P2YR的钙振荡。包括胶原蛋白、层粘连蛋白和纤连蛋白在内的包被底物不会影响P2YR的自发活性。P2YR细胞外精氨酸-甘氨酸-天冬氨酸（RGD）基序的突变会抑制自发活性。此外，依赖P2YR的自发钙振荡需要细胞外钙。GPCR筛选试验鉴定出表达10种GPCRs的细胞，包括嘌呤能P2YR、P2YR和P2YR，它们在细胞培养软底物下表现出自发钙振荡。我们的结果表明，细胞粘附表面的硬度通过一种不依赖配体的机制调节包括P2YR在内的几种GPCRs的自发活性。
