Regulation of Endothelial Cell function by Integration of Gαi and β-arrestin signaling at Atypical Chemokine Receptor 3

Atypical Chemokine receptor 3 (ACKR3), also known as C-X-C chemokine receptor type 7 (CXCR7), is a G protein-coupled receptor (GPCR) implicated in several physiological processes including leukocyte trafficking, cancer, and angiogenesis. Commonly co-expressed with C-X-C chemokine receptor type 4 (CXCR4), ACKR3 is classified as an β-arrestin-biased "scavenger" receptor due to its lack of G-protein signaling and its ability to efficiently internalize and degrade its ligands, CXCL11 and CXCL12. However, much is still unknown regarding ACKR3 signaling mechanisms and its implications on cellular physiology. Here we describe ongoing studies to characterize the mechanisms that underlie ACKR3 signaling and endothelial cell function. We demonstrated two ACKR3 specific agonists, WW36 and WW38, promote proliferation and migration of human umbilical vein endothelial cells (HUVECs). Surprisingly, we found that ACKR3-promoted proliferation and migration was sensitive to pertussis toxin (PTX), suggesting the involvement of Gαi/o proteins at a receptor which does not canonically signal via G proteins. We also observed similar sensitivity to PTX on ERK activation in HUVECs. We hypothesized that these results may in part be explained by the recently described coordination of Gαi and β-arrestin (βarr) to promote Gαi:βarr complex-specific downstream signaling. In support of this hypothesis, we demonstrated the formation of a Gαi:βarr complex upon ACKR3 activation in a dose-dependent manner. To characterize the necessary components in Gαi and βarr, we first developed active proximal polar core mutant and proximal finger loop βarr mutant and tested these at the prototypical class B Vasopressin 2 Receptor. Both mutants lead to considerable decrease in complex formation with the finger loop mutation nearly abolishing our split signal. Within the Gαi subfamily, we observed considerable differences in the ability to form complex, with Gαi2 and Gαi3 promoting less complex formation than Gαi1 and Gαo. We developed several Gαi mutants, hypothesizing that residues that differ between Gαi isoforms may impact Gαi:βarr formation. Using NanoBRET we demonstrated that these mutants impair complex formation, suggesting that they may be involved either via direct contacts with βarr or impacting Gαi conformation such that complex formation is no longer as favorable. These studies reveal a novel role for Gαi:βarr complexes in mediating ACKR3 function that contribute to endothelial cell biology. Additionally, these findings support an emerging paradigm in which G proteins and βarr coordinate their signaling downstream of GPCRs.

Duke Scholars

Author Sudarshan Rajagopal Medicine, Cardiology