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Highlights d ACKR3 function was analyzed in b-arrestinand phosphorylation-deficient conditions d Phosphorylation regulates ACKR3-mediated scavenging independently of b-arrestins d b-Arrestin signaling is dispensable for CXCL12 guidance of interneuron migration d Absence and excess of CXCL12 cause similar interneuron motility defects

Background: CXCR7 is an atypical heptahelical receptor that functions as scavenger for the endogenous chemokine ligand but fails to signal through G-proteins. Results: The CXCR7 C terminus causes uncoupling from G-protein, rapid receptor-mediated ligand uptake, and constitutive receptor degradation. Conclusion: Atypical CXCR7 functions are encoded in C-terminal elements. Significance: Identification of structural determinants regulating atypical and canonical functions of heptahelical receptors.

The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function.