Adaptor protein2 (AP2) orchestrates CXCR2-mediated cell migration

Abstract

The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, β2, μ2 and σ2 subunits, facilitates clathrin-mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl-terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand-mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand-mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2-μ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, μ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2-μ2, is replaced with μ2 mutated in Patch 1 and/or Patch 2 domains, ligand-mediated receptor binding and internalization are not lost. However, chemotaxis requires AP2-μ2 Patch 1, but not Patch 2. AP2-σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2-σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2-mediated receptor internalization can be dissociated from AP2-mediated chemotaxis. The dileucine motif of CXCR2 interacts with adaptor protein 2 (AP2) to facilitate ligand-mediated receptor internalization and chemotaxis. Using knockdown/rescue approaches involving mutations in AP2-μ2 and AP2-σ2 subunits involved in PIP2 binding versus receptor, respectively, we evaluated ligand-mediated receptor internalization, cell polarization and chemotaxis. Both electropositive Patch 1 amino acid residues on AP2-μ2 and the hydrophobic residues on AP2-σ2 are vital for directional migration and chemotaxis, while clathrin is dispensable. Importantly, AP2-mediated receptor internalization can be dissociated from AP2-mediated chemotaxis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.