Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and β-arrestin adaptor proteins. G protein- and β-arrestin-mediated signaling have been considered separable. We show GPCRs promote a direct interaction between Gαi protein subtype family members and β-arrestins, regardless of their canonical Gαi protein subtype coupling. Gαi:β-arrestin complexes bound extracellular signal-regulated kinase (ERK) and their disruption impaired both ERK activation and cell migration, consistent with β-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:β-arrestin signaling complexes.

47G-protein-coupled receptors (GPCRs) enable cells to sense and respond appropriately 48 to hormonal and environmental signals, and are a target of ~30% of all FDA-approved 49 medications. Canonically, each GPCR couples to distinct Gα proteins, such as Gαs, Gαi, Gαq or 50 Gα12/13, as well as b-arrestins. These transducer proteins translate and integrate 51 extracellular stimuli sensed by GPCRs into intracellular signals through what are broadly 52 considered separable signalling pathways. However, the ability of Gα proteins to directly interact 53 with b-arrestins to integrate signalling has not previously been appreciated. Here we show a 54 novel interaction between Gαi protein family members and β-arrestin. Gαi:β-arrestin complexes 55 were formed by all GPCRs tested, regardless of their canonical G protein isoform coupling, and 56 could bind both GPCRs as well as the extracellular signal-regulated kinase (ERK). This novel 57 paradigm of Gαi:β-arrestin scaffolds enhances our understanding of GPCR signalling. 58 93 protein:protein interactions 9 ) by complementing a small peptide (smBiT) fused to one protein to 94 a large protein fragment (LgBiT) fused to another protein of interest. The signal generated by 95 complementation of this split luciferase can then transfer to a third protein tagged with a 96 fluorescent protein acceptor, monomeric Kusabira Orange (mKO), generating a BRET 97 response. Thus, this technique enables real-time quantification of interactions between a two-98 protein complex and a third protein in living cells. 99Using this technology, we were surprised to discover that the canonically Gas-coupled V2R 100 also formed a 'megaplex' with Gai and β-arrestin following agonist treatment ( Fig. 1d,e). To further 101 interrogate the Gai:β-arrestin:V2R megaplex, we proceeded to swap the location of dipole donor 102 and acceptor components ( Fig. 1f). Altering the location of complex BRET components increased 103 the observed signal of the Gai-containing megaplex following agonist treatment, and further 104 confirmed that Gai:β-arrestin complexes can associate with the V2R (Fig 1g,h, Extended Data Fig. 105 1a). Agonist treatment of the canonically Gas-coupled β2AR also formed Gai:β-arrestin:β2AR 106 megaplexes (Fig 1i,j, Extended Data Fig. 1b), although less robustly than the V2R. We further 107 validated the specificity of megaplex formation by simultaneously transfecting both mKO-tagged 108 and untagged V2R and β2AR and treating with agonist. Only treating a mKO-tagged receptor with 109 its cognate agonist formed an observable Gai:β-arrestin:GPCR megaplex (Extended Data Fig 1c-110 h), indicating a specific interaction and not a bystander effect. 111 112 Figure 1: Formation of G protein:b-arrestin:GPCR megaplexes. a, Arrangement of luciferase fragments and mKO 113 acceptor fluorophore for complex BRET on G protein (LgBiT), b-arrestin (mKO), and V2R (smBiT). HEK 293T cells were 114 transiently transfected with the indicated receptor and assay components and stimulated with the indicated agonist or 115...

Visual Abstract

Some G protein-coupled receptor (GPCR) ligands act as “biased agonists” that preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the GPCR CXC chemokine receptor 3 (CXCR3). The signaling profile of CXCR3 changes as it traffics from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling is critical for biased activation of G proteins, β-arrestins, and extracellular-signal-regulated kinase (ERK). In CD8 + T cells, the chemokines promote unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation is dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.