β‐arrestin‐biased ACKR3 Promotes Gαi:β‐arrestin Complex Formation

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.

Duke Scholars

Author Sudarshan Rajagopal Medicine, Cardiology