AIP1 Functions as Arf6-GAP to Negatively Regulate TLR4 Signaling

Toll-like receptor 4 (TLR4) is unique among the Toll-like receptors in its ability to utilize TLR/IL1R-domain-containing adaptor protein (TIRAP), which recruits TLR4-MyD88 to phosphatidylinositol 4,5-bisphosphate (PIP2)-rich sites on the plasma membrane, to activate NF-kappa B and MAPK pathways. Here, we show that AIP1 disrupts formation of the TLR4-TIRAP-MyD88 complex without directly binding to any of the complex components. AIP1 via its pleckstrin homology and C2 domains binds to phosphatidylinositol 4-phosphate, a lipid precursor of PIP2. Knock-out of AIP1 in cells increases and overexpression of AIP1 reduces cellular PIP2 levels. We further show that AIP1 is a novel GTPase-activating protein (GAP) for Arf6, a small GTPase regulating cellular PIP2 production and formation of the TLR4-TIRAP-MyD88 complex. Thus, deletion of the GAPdomain on AIP1 results in a loss of its ability to mediate the inhibition of Arf6- and TLR4-induced signaling events. We conclude that AIP1 functions as a novel Arf6- GAP to negatively regulate PIP2-dependent TLR4-TIRAP-MyD88 signaling.