Investigation of the fate of type I angiotensin receptor after biased activation.

Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed β-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT1-R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged β-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, angiotensin II-stimulated wild-type AT1-R showed differences compared with a biased mutant AT1-R (DRY/AAY AT1-R) with regards to β-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the β-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor.

Szakadáti G ，Tóth AD ，Oláh I ，Erdélyi LS ，Balla T ，Várnai P ，Hunyady L ，Balla A ... -  《-》

Acute depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate impairs specific steps in endocytosis of the G-protein-coupled receptor.

Tóth DJ ，Tóth JT ，Gulyás G ，Balla A ，Balla T ，Hunyady L ，Várnai P ... -  《-》

Biased signaling of the angiotensin II type 1 receptor can be mediated through distinct mechanisms.

Bonde MM ，Hansen JT ，Sanni SJ ，Haunsø S ，Gammeltoft S ，Lyngsø C ，Hansen JL ... -  《PLoS One》

Allosteric modulation of β-arrestin-biased angiotensin II type 1 receptor signaling by membrane stretch.

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, β-arrestin, in a manner consistent with previously identified β-arrestin-biased ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-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 β-arrestin-activating conformations of the AT1R by using β-arrestin2-biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-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-β-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-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 β-arrestin-biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.

Tang W ，Strachan RT ，Lefkowitz RJ ，Rockman HA ... -  《-》

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors.

G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects. Using GPCR conformation-sensitive biosensors, we investigated allosterically induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer. Ligand occupancy of the AT1R induced distinct conformational changes in FP compared with those driven by PGF2α in bioluminescence resonance energy transfer (BRET)-based FP biosensors engineered with Renilla luciferase (RLuc) as an energy donor in the C-tail and fluorescein arsenical hairpin binder (FlAsH)-labeled acceptors at different positions in the intracellular loops. We also found that this allosteric communication is mediated through Gαq and may also involve proximal (phospholipase C) but not distal (protein kinase C) signaling partners. Interestingly, β-arrestin-biased AT1R agonists could also transmit a Gαq-dependent signal to FP without activation of downstream Gαq signaling. This transmission of information was specific to the AT1R/FP complex, as activation of Gαq by the oxytocin receptor did not recapitulate the same phenomenon. Finally, information flow was asymmetric in the sense that FP activation had negligible effects on AT1R-based conformational biosensors. The identification of partner-induced GPCR conformations may help identify novel allosteric effects when investigating multiprotein receptor signaling complexes.

Sleno R ，Devost D ，Pétrin D ，Zhang A ，Bourque K ，Shinjo Y ，Aoki J ，Inoue A ，Hébert TE ... -  《-》