Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors

WHIM syndrome is a rare congenital immunodeficiency often caused by mutations in the last 10-19 C terminal amino acids of CXCR4. These mutations impair CXCR4 internalization and increase responsiveness to CXCL12. The CXCR4 C-terminal domain also has a binding site for the actin-binding protein filamin A (FLNA); it is not known whether FLNA binds to WHIM CXCR4 mutants, or whether this interaction is implicated in the hyperfunction of these receptors. Here we show that, in addition to the CXCR4 C-tail, FLNA interacted with a region in the receptor third intracellular loop (ICL3) spanning amino acids 238-246. This interaction involved specific FLNA repeats and was sensitive to Rho kinase inhibition. Deletion of the 238-246 motif accelerated CXCL12-induced WT receptor endocytosis, but enabled CXCL12-mediated endocytosis and normalized signaling by the WHIM-associated receptor CXCR4 R334X . CXCL12 stimulation triggered CXCR4 R334X internalization in FLNA-deficient M2 cells but not in the FLNA-expressing M2 subclone A7; this suggests a role for FLNA in stabilization of WHIM-like CXCR4 at the cell surface. FLNA increased β-arrestin2 binding to CXCR4 R334X in vivo, which provides a molecular basis for FLNA-mediated hyperactivation of WHIM receptor signaling. We propose that FLNA interaction with ICL3 is central for endocytosis and signaling of WT and WHIM-like CXCR4 receptors.