GPCR kinase 3 phosphorylates 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 (GPCR) kinases (GRKs) to efficiently control chemokine levels. Despite not coupling G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. Here we resolve the underlying mechanisms allowing ACKR4 to circumvent the G protein requirement for GRK3 function. Using live cell BRET assays, we confirm that ACKR4 is preferentially phosphorylated by the GRK2/3 kinase family and that both GRK recruitment and receptor phosphorylation occur in the absence of activated G proteins. Instead, the kinases are recruited directly by a unique acidic rich motif in the proximal receptor C-terminus which coordinates productive phosphorylation reactions. Mutations in this region severely attenuated kinase recruitment and phosphorylation. Productive phosphorylation reaction plays a substantial role in the G protein-independent mechanism and a kinase-dead GRK3 (KD-GRK3) has severely reduced recruitment to ACKR4. This was not observed for KD-GRK3 translocation to GPCRs that recruit the kinase in a G protein-dependent manner. Together, these findings suggest that ACKR4 directly coordinates GRK3 recruitment and phosphorylation, highlighting a uniquely evolved atypical mechanism to utilize GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor.