G protein-coupled receptor kinase 3 couples atypical chemokine receptor 4 independent of G proteins.

Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein-coupled receptor kinases (GRKs) to efficiently control chemokine levels. Despite not coupling to G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. How ACKR4 circumvents G protein dependency to engage GRK3 is unclear. To resolve the underlying mechanism, we used live-cell bioluminescence resonance energy transfer assays to measure GRK3 and phosphorylation-dependent arrestin recruitment and tracked the impact of fluorescent chemokine uptake by flow cytometry. We demonstrate that ACKR4 engages arrestin preferentially downstream of GRK2/3 phosphorylation fully independent of G protein coordination. Instead, the kinases are recruited directly to the atypical receptor via a unique acidic-rich motif in the proximal receptor C terminus. Mutations in this region severely impaired kinase and arrestin recruitment, as well as chemokine scavenging. Productive phosphorylation also plays a substantial role in G protein-independent GRK3 translocation to ACKR4, and recruitment of kinase-dead GRK3 is severely impaired. Together, these findings suggest that ACKR4 directly coordinates GRK3 coupling, highlighting a uniquely evolved atypical mechanism to use GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor. SIGNIFICANCE STATEMENT: Cell migration and positioning is efficiently regulated by atypical chemokine receptors (ACKR) through chemokine scavenging, often upon GRK phosphorylation. GRK3 dominates the phosphorylation of ACKR4, despite ACKR4 not activating G proteins needed to promote the kinase activity. This study resolved that ACKR4 is directly modified by GRK3 without G protein involvement. Instead, specific acidic residues coordinate the phosphorylation reaction. While seemingly unique to ACKR4, similar mechanisms for GRK2/3 action may contribute to kinase modification of other atypical and canonical GPCRs.