title: Constitutive endocytosis of the chemokine CX3CL1 prevents its degradation by cell surface metalloproteases.
abstract: CX(3)CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX(3)CL1 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 CX(3)CL1 at the cell surface. We therefore investigated how transmembrane CX(3)CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX(3)CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX(3)CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX(3)CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX(3)CL1 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 CX(3)CL1. Although CX(3)CL1 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 CX(3)CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX(3)CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX(3)CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX(3)CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children Research Institute, Toronto M5G1X8, Canada.
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Huang
authors.forename: Paul
authors.initials: P
authors.lastname: Su
authors.forename: Guang Ying
authors.initials: GY
authors.lastname: Liu
authors.forename: Min Rui
authors.initials: MR
authors.lastname: Crow
authors.forename: Deanna
authors.initials: D
authors.lastname: Chaukos
authors.forename: Harry
authors.initials: H
authors.lastname: Yan
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Robinson
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D054428:Chemokine CX3CL1 / Q000235:genetics / Q000378:metabolism*; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D033962:Adaptor Protein Complex 2; C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D006835:Hydrazones; D008565:Membrane Proteins; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D034741:RNA, Small Interfering; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; D034281:Dynamins