2017/04/01 by Nathaniel G. dela Paz, Benoît Melchior, John A. Frangos
paper · doi:10.1152/ajpcell.00148.2016
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/Gα 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 Gα q/11 from its receptor S1P 3 , followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and β-arrestin-1/2 with S1P 3 in human coronary artery ECs, which are consistent with GPCR/Gα q/11 activation and receptor desensitization/internalization. The G protein activator AlF 4 resulted in increased dissociation of Gα q/11 from S1P 3 , but no increase in association between S1P 3 and either GRK2 or β-arrestin-1/2. The G protein inhibitor guanosine 5′-(β-thio) diphosphate (GDP-β-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 /Gα q/11 . There were no increased associations between S1P 3 and GRK2 or S1P 3 and β-arrestin-1/2 until 5 min. GDP-β-S, but not VPC23019, prevented dissociation of Gα q/11 from S1P 3 in response to shear stress. Shear stress did not induce rapid dephosphorylation of β-arrestin-1 or rapid internalization of S1P 3 , indicating no GPCR activation. These findings suggest that Gα q/11 participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.