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

Abstract

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and barrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Community guidelines for GPCR ligand bias: IUPHAR review 32

“…This extends the number of pathways to 27: 16 Gα proteins, 7 GRKs, and 4 arrestin proteins. For example, G proteins belonging to the same family may differ in their functional outcome due to unique binding kinetics, cellular expression levels, and engagement of different downstream effectors (Anderson et al, 2020;Avet et al, 2021;Ho & Wong, 2001;Jiang & Bajpayee, 2009;Olsen et al, 2020). Similarly, differential recruitment of the two isoforms of β-arrestin (β-arrestin 1-2) can translate to distinct functional outcomes, with respect to regulatory and signalling paradigms (Ghosh et al, 2019;Srivastava, Gupta, Gupta, & Shukla, 2015).…”

“…G q , G 11 , G 14 , and G 15 in the G q/11 family). Differential activation or recruitment of individual transducer family members has been shown both for G protein families (Avet et al, 2021;Inoue et al, 2019;Namkung et al, 2018;Olsen et al, 2020) and the arrestin family (Avet et al, 2021;Srivastava, Gupta, Gupta, & Shukla, 2015).…”

“…recent assays consistently profiling G proteins and arrestins (also with GRKs)(Avet et al, 2021;Olsen et al, 2020) (The assays in Olsen et al were optimized from Gales et al 2006; Lukasheva et al, 2020). b.…”

Nucleotide-decoupled G proteins reveal the role of G protein conformation in receptor-G protein selectivity

“…S2). As one example, M 4 R displayed robust agonist-induced BRET with 4A heterotrimers from all four G protein families, even though these receptors cannot activate wt G s/olf and G 12/13 heterotrimers and only weakly activate G q 20 . More stringent selectivity between 4A mutants was retained in a few receptors, and this subset included receptors that couple primarily to each of the four G protein families.…”

“…For example, the G s -coupled bile acid receptor GPBAR exhibited more stringent selectivity for G s 4A heterotrimers, associating with other G protein 4A mutants only in the presence of high concentrations of agonist. Notably, GPBAR is also highly selective for wt G s/olf over other heterotrimers 20 . Likewise, 5-HT 2A serotonin receptors and GPR55 retained fairly strict selectivity for their cognate heterotrimers G q 4A and G 13 4A, respectively.…”