Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases

CX3CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX3CL1 is the only chemokine known to undergo constitutive internalization, raising the possibility that dynamic equilibrium between the endocytic compartment and the plasma membrane critically regulates the availability and processing of CX3CL1 at the cell surface. We therefore investigated how transmembrane CX3CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX3CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX3CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX3CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX3CL1 lacking the cytoplasmic tail. Deletion of the cytosolic tail precluded internalization of the chemokine. We used site-directed mutagenesis to disrupt AP-2-binding motifs, singly or in combination, which resulted in diminished internalization of CX3CL1. Although CX3CL1 was present in both superficial and endomembrane compartments, ADAM10 (a disintegrin and metalloprotease 10) and tumor necrosis factor-converting enzyme, the two metalloproteases that cleave CX3CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX3CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX3CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX3CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.