Recycling of the Membrane-anchored Chemokine, CX3CL1

Abstract

CX 3 CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX 3 CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX 3 CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX 3 CL1 in human ECV-304 cells stably expressing untagged or green fluorescent proteintagged forms of the chemokine. CX 3 CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX 3 CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX 3 CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX 3 CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX 3 CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli.

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

“…The expression and the function of CX 3 CL1 in endothelial cells have been well described (6,7,27,43). In the kidney, an important role for CX 3 CL1 has been found in diseases that involve glomerular inflammation and endothelial injury (19,20,22,23).…”

“…Immunoblotting SDS-PAGE and immunoblotting were performed using anti-CX 3 CL1 Ab (0.2 g/ml) (27,34). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences UK Limited, Buckinghamshire, UK) recorded on x-ray film.…”

“…Primary human RTEC (Clonetics, Cambrex, Walkersville, MD) were a gift from Dr. Jim Scholey (University of Toronto) and were cultured according to the manufacturer's instructions (26). CX 3 CL1 expression in these cells was verified by Western blotting (27). Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized peripheral blood from healthy volunteers (28).…”

“…DNA constructs for CX 3 CL1 or CX 3 CL1 tagged with green fluorescent protein (CX 3 CL1-GFP) were created as described previously (6,27,29). MDCK cells were stably transfected with these constructs using FuGENE (Roche, Indianapolis, IN) and selected in 500 g/ml G418 (Wisent).…”

Transmembrane chemokines: Versatile ‘special agents’ in vascular inflammation

“…From hereCX3CL1 can be endocytosed and recycled to thecellmembrane (18).In cellc ulture experiments and in body fluids including blood serum, cerebrospinal and synovial fluid soluble forms of CX3CL1 and CXCL16 ared etectable (see below). To date,n o evidencet hat these forms arise from differential splicing has beenp rovided.I nstead, the conversion of transmembrane into solubleCX3CL1 and CXCL16has been attributedtoametalloproteolytic process (19,20).…”