Distinct conformational changes in β-arrestin report biased agonism at seven-transmembrane receptors

Abstract

␤-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct ␤-arrestin-mediated signaling. The recent discovery of ␤-arrestinbiased ligands and receptor mutants has allowed characterization of these independent ''G protein-mediated'' and ''␤-arrestinmediated'' signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functions of ␤-arrestins remain unclear. Here, using an intramolecular BRET (bioluminescence resonance energy transfer)-based biosensor of ␤-arrestin 2 and a combination of biased ligands and/or biased mutants of three different 7TMRs, we provide evidence that ␤-arrestin can adopt multiple ''active'' conformations. Surprisingly, phosphorylation-deficient mutants of the receptors are also capable of directing similar conformational changes in ␤-arrestin as is the wild-type receptor. This indicates that distinct receptor conformations induced and/or stabilized by different ligands can promote distinct and functionally specific conformations in ␤-arrestin even in the absence of receptor phosphorylation. Our data thus highlight another interesting aspect of 7TMR signaling-i.e., functionally specific receptor conformations can be translated to downstream effectors such as ␤-arrestins, thereby governing their functional specificity.7TMRs ͉ BRET ͉ phosphorylation G protein-coupled receptors (GPCRs), also known as seventransmembrane receptors (7TMRs), are the largest family of cell-surface receptors that communicate extracellular stimuli to the cell interior (1). The classical view of GPCR signaling is that a ligand activates the receptor and an activated receptor then couples to and activates heterotrimeric G proteins, leading to generation of second messengers such as cAMP, DAG, and IP3. Subsequently, G protein-coupled receptor kinases (GRKs) phosphorylate the receptor and promote ␤-arrestin recruitment to the receptor. Binding of ␤-arrestins to the receptor sterically inhibits further G protein coupling and leads to receptor desensitization.

Coarse-grained modeling of allosteric regulation in protein receptors

“…2E), our model identifies residues 113-115, 182-184, and 299-301 (located in the immediate environment of the retinal cofactor), residues 135-137, which nearly coincide with the ERY motif [residues 134-136 (40)], and residues 66-68, 225-227, 143-147, 235-245, and 312-323 (the eighth α-helix), which are also known to participate in G-protein binding (40). Our model also suggests that loop residues 278-283 at the periplasmic side may be involved in allosteric regulation by influencing ligand binding (47,48).…”

“…If the receptor mediates 2 or more signaling pathways, ligand binding could activate one pathway but suppress another. Indeed, selected activation of the "G protein-mediated" pathway or the "β-arrestin-mediated" pathway in B2AR (termed "biased agonism" or "functional selectivity") has been recently discovered in experiments (47,48).…”

GPCRs and G Protein Activation

“…This indicates that arrestin undergoes a significant conformation change when it recognizes the phosphorylated receptors. This is confirmed by the observation that the arrestin sensitivity to proteolytic degradation increases upon GPCR recognition, and that the intramolecular BRET between the N-and C-terminal region of a luciferase-arrestin-Yellow Fluorescent Protein (YFP) construct is markedly affected by arrestin recognition of agonist-bound receptors (Shukla et al, 2008).…”

Selective activation of the M 1 muscarinic acetylcholine receptor achieved by allosteric potentiation

“…5). Other studies show that AC-42 does not activate all G-proteins coupled to M 1 (35), together suggesting that these allosteric agonists may exhibit ligand bias (36), the propensity to activate a subset of signaling pathways presumably by stabilizing unique receptor conformations. Since ␤-arrestins couple M 1 to diacyl glycerol kinases (24) and likely play other signaling functions, it will be interesting to identify ␤-arrestin-dependent responses triggered by ACh in hippocampal neurons.…”