Illuminating G-Protein-Coupling Selectivity of GPCRs

Abstract

SUMMARY Heterotrimetic G proteins consist of four subfamilies (GS, Gi/O, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

“…Other assays relying on pathway-specific transcriptional activation of reporter genes (i.e., CRE, SRE, SRF-RE, NFAT-RE) (Cheng et al, 2010;Siehler, 2008) or engineered transcription-or proteolytically-activated reporter systems (i.e. PRESTO-TANGO (Kroeze et al, 2015) or TGF-α shedding assays (Inoue et al, 2019)) also involve amplification steps and long incubation times. Many of these relatively distal readouts rely on biological responses that can be modulated by multiple downstream signaling pathways and lead to cross-talk, complicating data interpretation (Mancini et al, 2015).…”

“…Second, our assays use native untagged GPCR and G protein subunits, contrary to complementation (i.e., split luciferase (Laschet et al, 2019), PRESTO-TANGO (Kroeze et al, 2015)), FRET/BRET-based Gα-Gβγ dissociation/receptor-G protein interaction (Bunemann et al, 2003;Gales et al, 2005;Gales et al, 2006;Hoffmann et al, 2005;Lohse et al, 2003;Mende et al, 2018;Namkung et al, 2018) or TGF-α shedding (Inoue et al, 2019) assays that require modification of receptors and/or G proteins. Modifying these core-signaling components could alter responses and complicate interpretation.…”

“…Globally, we assessed whether the receptor/G protein coupling profile similarities could be correlated with the Gα protein sequence identity using pairwise comparison ( Figure 5A). The comparison was performed using either the full-length G protein sequences or particular domains important for their activity including the H5 domain that penetrates into the receptor cavity, the 7 last amino acid of H5 domains used by Inoue et al to create their G protein chimeras for G protein profiling (Inoue et al, 2019) and the G protein barcode proposed by Flock et al as the determinant underlying selective G protein coupling (Flock et al, 2017). Reasonable correlations were observed in all cases with the highest correlation observed for the H5 domain and the G protein barcode (r 2 ≥ 0.80) and the lowest with 7 last amino acid of H5 (r 2 = 0.65).…”

“…We compared the signaling profiles that we observed using the EMTA platform with that of the chimeric G protein-based assay developed by Inoue et al (Inoue et al, 2019) and the Guide to Pharmacology database (GtoPdb; https://www.guidetopharmacology.org/). Among the 100 receptors that we tested, 71, 96 and 69 are shared with the Inoue, GtoPdb and both resources, respectively ( Figure 6A).…”

The influence of peptide context on signalling and trafficking of glucagon-like peptide-1 receptor biased agonists

“…Again, the Gly2-containing Phe1 ligands showed the greatest signalling deficit of all, with small magnitude responses even for Gs recruitment (which are, nevertheless, sufficient to produce full cAMP responses in the context of adequate amplification by adenylate cyclase). It should be noted that mini-G protein recruitment responses do not fully recapitulate G protein activation dynamics, which have recently been studied using NanoBRET-based sensors (43) and would be an interesting future investigation for these ligands. The β-arrestin-2 recruitment response of Chi2-phe1 was greater than that of other Phe1 agonists, which was not observed in the DiscoverX assay; this observation remains unexplained.…”