Research ArticleGPCR SIGNALING

G protein–coupled receptor kinases (GRKs) orchestrate biased agonism at the β2-adrenergic receptor

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Sci. Signal.  21 Aug 2018:
Vol. 11, Issue 544, eaar7084
DOI: 10.1126/scisignal.aar7084

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Determining bias

Biased signaling by GPCRs results in their preferential use of either G proteins or β-arrestins to transduce signals. Drugs that selectively activate one of these pathways provide effective treatment without side effects. Noting that GPCRs must undergo GRK-mediated phosphorylation to recruit β-arrestins, Choi et al. characterized a mutant GPCR that exhibits G protein–biased signaling and found that it could not be phosphorylated by GRKs. When a phosphorylated peptide was fused to the C-terminal region of the mutant receptor, mimicking GRK-mediated phosphorylation, β-arrestin protein recruitment and biased signaling were recovered. These data suggest that G protein–biased signaling is driven more by an inability to recruit GRKs than by an inability to couple to β-arrestins, which may have implications for the design of biased drugs.

Abstract

Biased agonists of G protein–coupled receptors (GPCRs), which selectively activate either G protein– or β-arrestin–mediated signaling pathways, are of major therapeutic interest because they have the potential to show improved efficacy and specificity as drugs. Efforts to understand the mechanistic basis of this phenomenon have focused on the hypothesis that G proteins and β-arrestins preferentially couple to distinct GPCR conformations. However, because GPCR kinase (GRK)–dependent receptor phosphorylation is a critical prerequisite for the recruitment of β-arrestins to most GPCRs, GRKs themselves may play an important role in establishing biased signaling. We showed that an alanine mutant of the highly conserved residue tyrosine 219 (Y219A) in transmembrane domain five of the β2-adrenergic receptor (β2AR) was incapable of β-arrestin recruitment, receptor internalization, and β-arrestin–mediated activation of extracellular signal–regulated kinase (ERK), whereas it retained the ability to signal through G protein. We found that the impaired β-arrestin recruitment in cells was due to reduced GRK-mediated phosphorylation of the β2AR Y219A C terminus, which was recapitulated in vitro with purified components. Furthermore, in vitro ligation of a synthetically phosphorylated peptide onto the C terminus of β2AR Y219A rescued both the initial recruitment of β-arrestin and its engagement with the intracellular core of the receptor. These data suggest that the Y219A mutation generates a G protein–biased state primarily by conformational selection against GRK coupling, rather than against β-arrestin. Together, these findings highlight the importance of GRKs in modulating the biased agonism of GPCRs.

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