Research Article

Noncanonical scaffolding of Gαi and β-arrestin by G protein–coupled receptors

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Science  12 Mar 2021:
Vol. 371, Issue 6534, eaay1833
DOI: 10.1126/science.aay1833

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Another way for GPCRs to signal

G protein–coupled receptors (GPCRs) normally transmit signals by coupling to heterotrimeric guanine nucleotide–binding proteins (G proteins) or by binding β-arrestin proteins. Smith et al. provide evidence for another mechanism, an approximate combination of the two. They monitored the interaction of vasopressin type 2 receptors (V2Rs) and Gα proteins in cultured cells using bioluminescent resonance energy transfer. Even though V2Rs do not signal canonically through Gαi proteins, they promoted the formation of complexes containing β-arrestin and Gαi, and this led to downstream signaling to extracellular signal-regulated kinase protein kinases.

Science, this issue p. eaay1833

Structured Abstract


G protein–coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning receptors and are the target of 30% of all U.S. Food and Drug Administration–approved medications. GPCRs are involved in nearly every human physiological process by serving as receptors for a wide array of ligands, including proteins, peptides, fatty acids, small molecules, and ions. The canonical signaling mechanisms of GPCRs involve coupling to specific G protein subtypes (e.g., Gαs, Gαi, Gαq, and Gα12) as well as β-arrestin adaptor proteins. Each G protein subtype regulates specific intracellular second messengers, such as cyclic adenosine 3′,5′-monophosphate (cAMP) responses or calcium mobilization. β-arrestins inhibit, or “arrest,” this canonical G protein–mediated signaling while also promoting their own intracellular signaling events such as extracellular signal–regulated kinase (ERK) activity. Because G proteins and β-arrestins can differentially regulate signaling pathways, often with distinct cellular effects, efforts are under way to design drugs that preferentially target either G protein or β-arrestin signaling to generate more selective GPCR-targeted drugs, i.e., biased ligands. In theory, biased ligands can improve the desired pharmacological properties of drugs while also minimizing their on-target side effects. However, potential coordination between G protein and β-arrestin signaling could have a significant impact on our fundamental understanding of GPCR signaling and the development of biased ligands.


G protein and β-arrestin signaling have broadly been considered separable intracellular pathways with distinct signal transduction mechanisms. However, several lines of evidence have also suggested the potential for coordination between G protein and β-arrestin signaling. For example, some GPCRs can simultaneously bind a G protein and a β-arrestin. Furthermore, when GPCRs were activated in cells lacking functional G proteins, some β-arrestin–mediated effects of GPCR activation were also absent. This could be interpreted as a role for G proteins in what had previously been considered solely β-arrestin–mediated cellular responses. To gain further insight into these experimental findings, we investigated whether there is a GPCR-signaling pathway that involves direct interactions between G proteins and β-arrestins.


We first developed a bioluminescent resonance energy transfer (BRET) approach to monitor tripartite protein interactions among GPCRs, G proteins, and β-arrestin. After confirming that we could monitor such interactions, we found that GPCRs can catalyze a direct interaction between the Gαi protein subtype and β-arrestins at the plasma membrane, and confirmed the interaction using a variety of biochemical and biophysical techniques. This interaction was only observed between β-arrestin and the Gαi protein family, not other Gα protein subtypes. These Gαi:β-arrestin complexes were formed downstream of all receptors tested, even with receptors that do not canonically signal through Gαi, such as the Gαs-coupled β2-adrenergic and vasopressin type 2 receptors. We found that Gαi:β-arrestin complexes formed scaffolds with the signaling kinase ERK downstream of certain receptors. Stimulation of the angiotensin type II receptor with a β-arrestin–biased agonist promoted the formation of the Gαi:β-arrestin complex despite not activating canonical G protein signaling. Cellular migration promoted by this β-arrestin–biased agonist was sensitive to inhibition of both β-arrestins and Gαi, which is consistent with Gαi:β-arrestin complexes regulating cellular migration.


Our results reveal a GPCR-signaling paradigm in which GPCRs promote the formation of Gαi:β-arrestin signaling complexes that are distinct from other canonical forms of GPCR signaling. These Gαi:β-arrestin complexes have the ability to scaffold important cellular effectors such as ERK, and can play a functional role in cellular responses such as cell migration. Demonstration of Noncanonical coordination of Gαi and β-arrestins by GPCRs adds to our understanding of the fundamental mechanisms underlying GPCR signaling, and the potential effects of this complex should be considered when designing and evaluating GPCR ligands.

GPCR signaling by Gαi:β-arrestin complexes.

Complementation and proximity assays identified a complex between Gαi protein family members, but not other Gα protein subtypes, and β-arrestin. This was observed across multiple receptors, including those that do not canonically couple to Gαi. This complex was associated with functional effects, including cell migration. These findings demonstrate GPCR signaling by Gαi:β-arrestin complexes.


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–mediated signaling and β-arrestin–mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between Gαi protein subtype family members and β-arrestins regardless of their canonical Gα protein subtype coupling. Gαi:β-arrestin complexes bound extracellular signal–regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is 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.

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