Phosphoproteomic mapping of CCR5 and ACKR2 signaling properties.

ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3 min) and long (30 min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.

Control of Cytoskeletal Dynamics by ß-Arrestin1/Myosin Vb Signaling Regulates Endosomal Sorting and Scavenging Activity of the Atypical Chemokine Receptor ACKR2.

The atypical chemokine receptor ACKR2, formerly named D6, is a scavenger chemokine receptor with a non-redundant role in the control of inflammation and immunity. The scavenging activity of ACKR2 depends on its trafficking properties, which require actin cytoskeleton rearrangements downstream of a ß-arrestin1-Rac1-PAK1-LIMK1-cofilin-dependent signaling pathway. We here demonstrate that in basal conditions, ACKR2 trafficking properties require intact actin and microtubules networks. The dynamic turnover of actin filaments is required to sustain ACKR2 constitutive endocytosis, while both actin and microtubule networks are involved in processes regulating ACKR2 constitutive sorting to rapid, Rab4-dependent and slow, Rab11-dependent recycling pathways, respectively. After chemokine engagement, ACKR2 requires myosin Vb activity to promote its trafficking from Rab11-positive recycling endosomes to the plasma membrane, which sustains its scavenging activity. Other than cofilin phosphorylation, induction of the ß-arrestin1-dependent signaling pathway by ACKR2 agonists also leads to the rearrangement of microtubules, which is required to support the myosin Vb-dependent ACKR2 upregulation and its scavenging properties. Disruption of the actin-based cytoskeleton by the apoptosis-inducing agent staurosporine results in impaired ACKR2 internalization and chemokine degradation that is consistent with the emerging scavenging-independent activity of the receptor in apoptotic neutrophils instrumental for promoting efficient efferocytosis during the resolution of inflammation. In conclusion, we provide evidence that ACKR2 activates a ß-arrestin1-dependent signaling pathway, triggering both the actin and the microtubule cytoskeletal networks, which control its trafficking and scavenger properties.

Liprin-α1 and ERC1 control cell edge dynamics by promoting focal adhesion turnover.

Liprin-α1 and ERC1 are interacting scaffold proteins regulating the motility of normal and tumor cells. They act as part of plasma membrane-associated platforms at the edge of motile cells to promote protrusion by largely unknown mechanisms. Here we identify an amino-terminal region of the liprin-α1 protein (liprin-N) that is sufficient and necessary for the interaction with other liprin-α1 molecules. Similar to liprin-α1 or ERC1 silencing, expression of the liprin-N negatively affects tumor cell motility and extracellular matrix invasion, acting as a dominant negative by interacting with endogenous liprin-α1 and causing the displacement of the endogenous ERC1 protein from the cell edge. Interfering with the localization of ERC1 at the cell edge inhibits the disassembly of focal adhesions, impairing protrusion. Liprin-α1 and ERC1 proteins colocalize with active integrin ß1 clusters distinct from those colocalizing with cytoplasmic focal adhesion proteins, and influence the localization of peripheral Rab7-positive endosomes. We propose that liprin-α1 and ERC1 promote protrusion by displacing cytoplasmic adhesion components to favour active integrin internalization into Rab7-positive endosomes.