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 B2-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.conformational transition ͉ ligand-independent activation ͉ mechanochemical signal transduction ͉ plasma membrane ͉ bradykinin

Flow enhances the agonist-induced responses of the bradykinin receptor (B2) because of its lectinic nature: Role of its oligosaccharide environment

“…Obviously, there is a need for a simpler model, where flow and the cell surface can be directly accessed, such as isolated blood vessels perfused at a controlled flow [7,8]. In this study, in the guinea pig carotid artery, the effects of flow on the bradykinin B2 receptor; which is directly activated by flow [1], were resolved using alterations of the vasoactive bradykinin effects which also altered upon an irreversible binding of an oligosaccharide polymer to the endothelial surface layer or the enzymatic removal of heparinic groups.…”

“…Interestingly, the roles of individual endothelial surface sugars and lectins in flow-induced responses are function biased; a complexity that makes difficult to identify the exact "mechanosensor(s)" for each response [6,8,[10][11][12][13]15]. In contrast, in the case of a defined G-protein coupled receptor, the response triggered by its specific agonist identifies it as the structure of origin, the agonist-sensor [1,3,4,14]. If this agonist-sensor induced response is modulated by flow, it would suggest that this G-protein coupled receptor is also a flow-sensor and because of being lectinic, this flow-modulated response will react to specific alterations of the endothelial surface layer oligosaccharide composition, suggesting that the oligosaccharide environment modulates the G-protein coupled receptor flow-sensitivity.…”

“…The molecular and physical mechanisms by which endothelial cells transduce flow into biochemical responses are ambiguous [1][2][3][4][5], because the transducing molecules directly affected by flow, the "mechanosensors", are unknown. Flow sensing properties likely reside in the luminal endothelial surface; constituted by the cell membrane and glycocalyx which is acted by flow [6][7][8][9][10][11][12][13].…”

“…Our hypothesis is that the flow response requires that the sensor, composed of a lectinic transducing protein in a specific oligosaccharide environment, detects flow/mechanical stress through lectin-oligosaccharide interactions [1,2,8,12,14,26,51]. This concept would apply to other flow-sensitive luminal endothelial membrane protein structures listed in Table 3.…”

“…Lastly, it is inaccurate to assume that hormone and electrical stimuli act on transmembrane effector proteins in a cell membrane (a lipid bilayer) which is denuded of extracellular specific anchor structures i. e. a "baldheaded" membrane [1,2,46]. These pervasive models are a conceptual oversimplification since electron microscopic, biochemical, and now functional studies show that many of these transmembrane proteins are lectins and are surrounded and bound to a dense mesh of specific oligosaccharides complexes.…”