Shear stress induces Gαq/11activation 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/Gαq/11pair in EC shear stress-induced mechanotransduction. We demonstrated that sphingosine 1-phosphate (S1P) stimulation causes a rapid dissociation at 0.5 min of Gαq/11from its receptor S1P3, followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and β-arrestin-1/2 with S1P3in human coronary artery ECs, which are consistent with GPCR/Gαq/11activation and receptor desensitization/internalization. The G protein activator AlF4resulted in increased dissociation of Gαq/11from S1P3, but no increase in association between S1P3and either GRK2 or β-arrestin-1/2. The G protein inhibitor guanosine 5'-(β-thio) diphosphate (GDP-β-S) and the S1P3antagonist VPC23019 both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 s of S1P3/Gαq/11There were no increased associations between S1P3and GRK2 or S1P3and β-arrestin-1/2 until 5 min. GDP-β-S, but not VPC23019, prevented dissociation of Gαq/11from S1P3in response to shear stress. Shear stress did not induce rapid dephosphorylation of β-arrestin-1 or rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gαq/11participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.
La Jolla Bioengineering Institute, La Jolla, California ndelapaz@ljbi.org.
Nathaniel G
NG
Dela Paz
La Jolla Bioengineering Institute, La Jolla, California.
Benoît
B
Melchior
La Jolla Bioengineering Institute, La Jolla, California.
John A
JA
Frangos
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism*; D008297:Male; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D049349:Receptors, Lysosphingolipid; D033081:Shear Strength / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000081025:Sphingosine-1-Phosphate Receptors; D013314:Stress, Mechanical
D008246:Lysophospholipids; D043562:Receptors, G-Protein-Coupled; D049349:Receptors, Lysosphingolipid; D000081025:Sphingosine-1-Phosphate Receptors; C568792:S1pr4 protein, mouse; C060506:sphingosine 1-phosphate; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D013110:Sphingosine
G protein-coupled receptors; endothelial cell; heterotrimeric G proteins; shear stress