# Ligand-Independent Spontaneous Activation of Purinergic P2Y6 Receptor Under Cell Culture Soft Substrate

## TL;DR

This study shows that the P2Y6 receptor can activate without a ligand when cells are cultured on soft silicon substrates, suggesting that substrate stiffness affects GPCR activity.

## Contribution

The study reveals a ligand-independent mechanism by which substrate stiffness modulates GPCR spontaneous activity.

## Key findings

- P2Y6R induces Ca2+ oscillation on silicon substrates without a nucleotide ligand.
- RGD motif mutation and absence of extracellular Ca2+ inhibit this spontaneous activity.
- Ten GPCRs, including P2Y1R, P2Y2R, and P2Y6R, show spontaneous activity on soft substrates.

## 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 P2Y6 receptor (P2Y6R) induced spontaneous Ca2+ oscillation without a nucleotide ligand when cells were cultured in a silicon chamber. This P2Y6R-dependent Ca2+ oscillation was absent in cells cultured in glass dishes. Coating substrates, including collagen, laminin, and fibronectin, did not affect the P2Y6R spontaneous activity. Mutation of the extracellular Arg-Gly-Asp (RGD) motif of P2Y6R inhibited spontaneous activity. Additionally, extracellular Ca2+ was required for P2Y6R-dependent spontaneous Ca2+ oscillation. The GPCR screening assay identified cells expressing 10 GPCRs, including purinergic P2Y1R, P2Y2R, and P2Y6R, that exhibited spontaneous Ca2+ oscillation under cell culture soft substrate. Our results suggest that stiffness of the cell adhesion surface modulates spontaneous activities of several GPCRs, including P2Y6R, through a ligand-independent mechanism.