G protein-coupled receptors sense fluid shear stress in endothelial cells

Abstract

Hemodynamic shear stress stimulates a number of intracellular events that both regulate vessel structure and influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established. Here, we show that mechanical perturbation of the plasma membrane leads to ligand-independent conformational transitions in a G protein-coupled receptor (GPCR). By using time-resolved fluorescence microscopy and GPCR conformation-sensitive FRET we found that stimulation of endothelial cells with fluid shear stress, hypotonic stress, or membrane fluidizing agent leads to a significant increase in activity of bradykinin B2 GPCR in endothelial cells. The GPCR conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single endothelial cell under fluid shear stress in real time. We show that this response can be blocked by a B(2)-selective antagonist. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of GPCRs. Therefore, we suggest that GPCRs are involved in mediating primary mechanochemical signal transduction in endothelial cells. We anticipate our experiments to be a starting point for more sophisticated studies of the effects of changes in lipid bilayer environment on GPCR conformational dynamics. Furthermore, because GPCRs are a major target of drug development, a detailed characterization of mechanochemical signaling via the GPCR pathway will be relevant for the development of new antiatherosclerosis drugs.

Fig. 1.

Detection of B₂ conformational change. (a) Integrated polarized fluorescence spectra of B2K chameleon in a single BAEC before and after stimulation with bradykinin. (b) Fluorescence microscopy image of BAECs expressing B2K chameleon. Image was taken through a CFP filter. (Magnification: × 650.) (c) Polarized fluorescence decay kinetics of B2K chameleon from a single BAEC before and after stimulation with agonist. (d) Response of FRET ratio of B2K chameleon in a BAEC to stimulation by different concentrations of bradykinin at t = 0.

Fig. 2.

Agonist-induced changes in fluorescence anisotropy. (a) Parallel and perpendicular components of integrated fluorescence spectrum of B2K chameleon in a BAEC before and after stimulation with bradykinin. (b) Fluorescence anisotropy spectra of B2K chameleon in a BAEC before and after stimulation with agonist. (c) Time-resolved emission anisotropy of B2K chameleon in a single BAEC before and after exposure to agonist. Transient anisotropy traces are shown at both CFP and YFP emission wavelengths. The fast decay of the YFP anisotropy (decay constant ≈1 ns) is a direct manifestation of the FRET process from CFP.

Fig. 3.

Response of B₂ GPCR to fluid shear stress. (a) Integrated polarized fluorescence spectra of B2K chameleon in a single BAEC before and after stimulation with fluid shear stress for 2 min. (b) Response of fluorescence decay kinetics of B2K chameleon to stimulation by shear stress for 2 min. (c) Response of FRET ratio of B2K chameleon in a BAEC exposed to shear stress in the absence and presence of selective B₂ antagonist. Flow is turned on at time t = 0 and off at t = 150 s. (d) Fluid shear stress induced change in FRET ratio of B2K chameleon in a BAEC as a function of shear stress magnitude.

Fig. 4.

Response of B₂ GPCR to hypotonic shock (a and b) and the membrane fluidizing agent benzyl alcohol (c and d). (a) Changes in integrated polarized fluorescence spectra of B2K chameleon in a single BAEC caused by stimulation by hypotonic shock for 15 min. (Inset) Time-dependent response of FRET ratio caused by hypotonic shock. (b) Polarized fluorescence decay kinetics of B2K chameleon from a BAEC before and after stimulation with hypotonic medium for 15 min. Increase in a fluorescence lifetime of the donor (CFP) and decrease in a fluorescence lifetime of the acceptor (YFP) are clearly visible. (c) The dynamics of FRET ratio of B2K chameleon in a BAEC exposed to benzyl alcohol at time t = 0. Gray line shows a fit with a monoexponential rise function with a time constant of 180 s. (d) Integrated fluorescence signal from a BAEC stained with molecular rotor KW-04 and exposed to benzyl alcohol at time t = 0.