Duke University Durham, North Carolina, United States
Taylor Kohlmann (Duke University)| Claudia Lee (Duke University)| Sudarshan Rajagopal (Duke University, Duke University)
Atypical chemokine receptor 3 (ACKR3)—previously known as CXC-chemokine receptor 7 (CXCR7)—is involved in a wide range of physiological processes including angiogenesis, neuronal development, and tumorigenesis. As a β-arrestin biased G protein-coupled receptor (GPCR), ACKR3 recruits β-arrestin, but lacks G protein activity. Work from our lab has demonstrated that Gαi and β-arrestin can form complexes together downstream of receptor stimulation, even upon activation by β-arrestin-biased agonists. Therefore, we hypothesized that stimulation of β-arrestin biased receptors such as ACKR3 also promote Gαi:β-arrestin complex formation. Our early results indicated that Gαi and β-arrestin 2 associate at ACKR3 when treated with the synthetic agonists WW36 and WW38. Here, we expanded our panel of ligands to include proenkephalin-derived BAM22 and endogenous chemokine CXCL11 in addition to WW36 and WW38, and we sought to characterize the canonical behavior of ACKR3 and assess its capacity to similarly promote Gαi:β-arrestin complex formation with endogenous ligands. Using TRUPATH as well as other bioluminescence resonance energy transfer (BRET)-based assays, we show that all ligands did not activate Gαi, while they stimulated dose-dependent β-arrestin 2 recruitment to ACKR3 and internalization. This aligned with the expected behavior of a β-arrestin-biased GPCR. Furthermore, using split-luciferase assays, we found that a Gαi:β-arrestin 2 complex consistently formed in a dose-dependent manner across all four distinct ligands. Further studies will be necessary to elucidate the mechanism of formation and functional significance of this Gαi:β-arrestin 2 complex in ACKR3 signaling.