Deciphering biased-agonism complexity reveals a new active AT1 receptor entity

Abstract

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but β-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling

“…We also confirmed that the absence of G 12 stimulation was not due to a non-functioning G 12 biosensor. Indeed, using the same experimental conditions with HEK293T cells co-expressing the thromboxane A 2 ␣ subtype (TP␣) receptor and the G 12 sensor, stimulation with the agonist U46619 triggering a decrease of BRET signal was easily detected as already described (32). This indicates that the G 12 biosensor is also functional, suggesting that GHS-R1a is not coupled to G 12 (Fig.…”

“…We paid particular attention to the selectivity of ligands toward a panel of G protein subtypes thanks to G protein activation biosensors that were recently developed (31,32). As previously reported, GHS-R1a displays one of the highest constitutive activity (24,26) in the GPCR family (45,46).…”

“…G Protein Activation BRET Assay-G protein activation was measured with the BRET assay previously described (31,32). Briefly, HEK293T cells grown in 10-cm culture dishes were cotransfected by Lipofectamine 2000 with GHS-R1a and G protein subunits (Rluc8-␣, -␤1, and ␥2-GF10).…”

“…Study with G Protein Activation BRET Biosensors-To test GHS-R1a ligands on the activation of different G protein subtypes besides G q , we then used a BRET 2 -based assay that monitors conformational changes of G proteins upon activation (31,32). This BRET assay measured a BRET signal between Rluc8 fused to the ␣ subunit and GFP10 fused to the ␤2 subunit, in an Rluc8-␣, -␤1, and ␥2-GFP10 complex (see under "Experimental Procedures").…”

“…In this context, we investigated here the selectivity of a panel of GHS-R1a synthetic ligands toward arrestin and G proteindependent pathways. We paid particular attention to the selectivity of ligands toward activation of several G protein subtypes and isoforms thanks to the use of recently developed G protein BRET-based biosensors that were recently developed (31,32). Our data suggest that some synthetic GHS-R1a ligands are selective G q agonists.…”