Structure of the M2 muscarinic receptor–β-arrestin complex in a lipid nanodisc

Abstract

Summary Following agonist activation, G protein-coupled receptors (GPCRs) recruit β-arrestin, which desensitizes heterotrimeric G protein signaling and promotes receptor endocytosis 1 . Additionally, β-arrestin directly regulates many cell signaling pathways that can induce cellular responses distinct from that of G proteins 2 . Here we present a cryo-electron microscopy (cryoEM) structure of β-arrestin1 (βarr1) in complex with muscarinic acetylcholine-2-receptor (M2R) reconstituted in lipid nanodiscs. The M2R-βarr1 structure shows a multimodal network of flexible interactions, including binding of the βarr1 N-domain to phosphorylated receptor residues and βarr1 finger loop insertion into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric G o protein structure 3 . Moreover, the cryoEM map reveals that the βarr1 C-domain edge engages the lipid bilayer. Through atomistic simulations, biophysical, biochemical, and cellular assays, we show that the C-edge is critical for stable complex formation, βarr1 recruitment, receptor internalization, and desensitization of G protein activation. Taken together, these data suggest the cooperative interactions of β-arrestin with both the receptor and phospholipid bilayer contribute to its functional versatility.

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

“…Nevertheless, it is tempting to hypothesize that other residue combinations bias arrestin-mediated signaling in this system towards different effectors (7,8). This is consistent with reported different shapes of the arrestin-receptor complex (68)(69)(70). We also identified different effects of G protein on this process, with Gs enhancing ERK1/2 but not Src activation.…”

“…Crystal structures of the receptor-arrestin complexes indeed reveal that arrestin binds to both the phosphorylated elements in the C-terminus and the pocket unique to active receptor (the main G protein binding site) (67)(68)(69)(70). While phosphorylation commonly reduces G protein coupling (71)(72)(73), in some cases it changes the G protein subtype that couples to receptor (50,74).…”

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“…A pioneering example was the determination of a structure of agonist-activated MOP-R in complex with Gi (Koehl et al, 2018). Recently, cryo-electron microscopy was used successfully for structural determination of agonist-activated GPCRs in association with β-arrestin, a physiologically important complex that has eluded conventional xray crystallography (Zhou et al, 2016;Yin et al, 2019;Huang et al, 2020;Staus et al, 2020). It has also enabled structural determination of a GPCR bound to both β-arrestin and G protein simultaneously, a complex which has been proposed to form on the endosome membrane and transduce a sustained form of signaling by certain GPCRs after they undergo ligand-induced internalization (Vilardaga et al, 2012;Nguyen et al, 2019).…”

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“…However, our mutational analysis suggests that this complex involves, at minimum, the phosphorylated pCT of SMO and the N-tail domain of PKA-C. Membrane lipid interactions may also contribute to these complexes, as the N-tail is myristoylated which can increase PKA-C membrane association in some settings (Bastidas et al, 2012;Gaffarogullari et al, 2011;Tillo et al, 2017;Zhang et al, 2015b). Intriguingly, recent structures of non-SMO GPCRs in complex with βarrestins have also revealed critical interactions with the receptor's phosphorylated cytoplasmic tail and lipids in the surrounding membrane (Huang et al, 2020;Staus et al, 2020). Thus, distinct GPCRs may engage a diverse set of downstream effectors, such as βarrestins or PKA-C, using similar structural principles.…”