Abstract
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.
Highlights
Gprotein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways
Ligand occupancy of the angiotensin II type 1 receptor (AT1R) induced distinct conformational changes in F prostanoid (FP) compared with those driven by PGF2␣ 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
When the cells were stimulated with angiotensin II (Ang II), we observed a change in the BRET across all the FP conformational biosensors in the panel but opposite in direction, suggesting that activation of the heterodimer partner caused a distinct conformational change in FP as compared with its cognate agonist (Fig. 1B)
Summary
To investigate the interplay between the protomers in the FP/AT1R heterodimer, we began by co-expressing each of our previously published FP-ICL3-RLucII conformational biosensors (FlAsH “walked” through the third intracellular loop (ICL3) at 5 different positions [39]) with wild-type AT1R (AT1R-WT) in HEK 293 cells In this configuration, the assay strictly reports on conformational rearrangements between different vantage points in ICL3 and the C terminus of FP induced in response to ligand stimulation, i.e. only changes in FP conformation are reported. As with direct stimulation of the conformational biosensor with PGF2␣, the FP ICL3 P4 sensor reported the largest ⌬BRET in response to Ang II and the response was dosedependent (supplemental Fig. 1B).
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