Abstract
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(6) receptor (P2Y(6)R) induced spontaneous Ca(2+) oscillation without a nucleotide ligand when cells were cultured in a silicon chamber. This P2Y(6)R-dependent Ca(2+) oscillation was absent in cells cultured in glass dishes. Coating substrates, including collagen, laminin, and fibronectin, did not affect the P2Y(6)R spontaneous activity. Mutation of the extracellular Arg-Gly-Asp (RGD) motif of P2Y(6)R inhibited spontaneous activity. Additionally, extracellular Ca(2+) was required for P2Y(6)R-dependent spontaneous Ca(2+) oscillation. The GPCR screening assay identified cells expressing 10 GPCRs, including purinergic P2Y(1)R, P2Y(2)R, and P2Y(6)R, that exhibited spontaneous Ca(2+) oscillation under cell culture soft substrate. Our results suggest that stiffness of the cell adhesion surface modulates spontaneous activities of several GPCRs, including P2Y(6)R, through a ligand-independent mechanism.