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.Inflammation is marked by the migration of circulating leukocytes into sites of injury, a process that occurs via a series of coordinated interactions between leukocytes and endothelial or epithelial cells. Central to this process are chemokines, a family of low molecular weight proteins that can attract leukocytes bearing the complementary receptors. When engagement of the chemokine receptor occurs, the leukocyte becomes activated and is induced to firmly adhere to the inflamed endothelium. These initial steps culminate in diapedesis of the leukocyte across the endothelium and migration into the injured tissue. The local complement of chemokines elaborated is organ-specific and varies with the type of inflammation present. In addition, specific leukocyte subsets also bear distinct chemokine receptors. In this way, chemokines and chemokine receptors confer organ specificity to leukocyte migration and help to “fine-tune” the nature of the observed inflammatory response.Among the 40 chemokines identified so far, CX₃CL1 is one of only two that have a transmembrane structure (1, 2). The chemokine domain of CX₃CL1 binds to its complementary receptor, CX₃CR1, through two distinct amino acid residues (3). The mucin stalk of CX₃CL1 allows efficient presentation of the chemokine to circulating leukocytes that express CX₃CR1, thereby allowing these leukocytes to be captured by the underlying endothelium (4, 5). CX₃CL1 also possesses a cytoplasmic tail 37 amino acids in length. However, the specific functions of the cytoplasmic tail have been left completely unexplored.Accumulating evidence demonstrates a critical role for CX₃CL1 in the pathogenesis of diverse inflammatory diseases, including atherosclerosis, systemic lupus erythematosus, and rejection of transplanted organs (6–15). Cell surface expression of CX₃CL1 is known to be regulated by proteolytic cleavage, or shedding, from the plasma membrane (16–18). Constitutive cleavage of CX₃CL1 occurs at low levels and is mediated by ADAM10 (a disintegrin and metalloprotease 10) (17). In response to inflammatory stimulation with lipopolysaccharide or to protein kinase C activation using phorbol 12-myristate 13-acetate, proteolytic cleavage of CX₃CL1 is markedly enhanced. Inducible cleavage of CX₃CL1 is mediated by tumor necrosis factor-α converting enzyme (TACE; ADAM17),² a related protease of the metzincin family (16, 18).In addition to proteolytic cleavage, surface expression of CX₃CL1 is also regulated by subcellular trafficking. We recently demonstrated that cell surface CX₃CL1 rapidly recycles to and from a specialized endocytic compartment, raising the possibility that the intracellular pool serves as a storage depot and that dynamic equilibrium between the endocytic compartment and the plasma membrane determines the availability and processing of transmembrane CX₃CL1 (19). In the current study, we explored whether the unique cytoplasmic tail of CX₃CL1 is important for this novel mode of regulation of the chemokine and whether it affects susceptibility of the chemokine to surface proteases. Our data suggest that plasmalemmal CX₃CL1 undergoes constitutive clathrin-mediated endocytosis (CME), facilitating storage of an intracellular pool of chemokine that is protected from cell surface metalloproteases.