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摘要
It has recently been appreciated that the angiotensin II type 1 receptor (AT1R), a prototypic member of the G protein-coupled receptor superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, beta-arrestin, in a manner consistent with previously identified beta-arrestin-biased ligands. However, the ligand-independent mechanism by which mechanical stretch promotes beta-arrestin-biased signaling remains unknown. Implicit in the concept of biased agonism (i.e. the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined whether mechanical stretch stabilizes distinct beta-arrestin-activating conformations of the AT1R by using beta-arrestin2-biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to beta-arrestin (AT1R-beta-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the beta-arrestin-biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-beta-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-G(q) fusion. Biophysical experiments with an intramolecular BRET beta-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize beta-arrestin2 active conformations that differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific beta-arrestin-biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.
- 出版日期2014-10-10
