Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells

Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/G alpha(q/11) pair in EC shear stress-induced mechanotransduction. We demonstrated that sphingosine 1-phosphate (S1P) stimulation causes a rapid dissociation at 0.5 min of G alpha(q/11) from its receptor S1P(3), followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and beta-arrestin-1/2 with S1P(3) in human coronary artery ECs, which are consistent with GPCR/G alpha(q/11) activation and receptor desensitization/internalization. The G protein activator A1F(4) resulted in increased dissociation of G alpha(q/11) from S1P(3), but no increase in association between S1P(3) and either GRK2 or beta-arrestin-1/2. The G protein inhibitor guanosine 5'-(beta-thio) diphosphate (GDP-beta-S) and the S1P(3) antagonist VPC23019 both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 s of S1P(3)/G alpha(q/11). There were no increased associations between S1P(3) and GRK2 or S1P(3) and beta-arrestin-1/2 until 5 min. GDP-beta-S, but not VPC23019, prevented dissociation of G alpha(q/11) from S1P(3) in response to shear stress. Shear stress did not induce rapid dephosphorylation of beta-arrestin-1 or rapid internalization of S1P(3), indicating no GPCR activation. These findings suggest that G alpha(q/11) participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.