CX3CR1 tyrosine sulfation enhances fractalkine-induced cell adhesion.
Fractalkine is a unique CX(3)C chemokine/mucin hybrid molecule that functions like selectins in inducing the capture of receptor-expressing cells. Because of the importance of tyrosine sulfation for ligand binding of the selectin ligand PSGL1, we tested the role of tyrosine sulfation for CX(3)CR1 function in cell adhesion. Tyrosine residues 14 and 22 in the N terminus of CX(3)CR1 were mutated to phenylalanine and stably expressed on K562 cells. Cells expressing CX(3)CR1-Y14F were competent in signal transduction but defective in capture by and firm adhesion to immobilized fractalkine under physiologic flow conditions. In static binding assays, CX(3)CR1-Y14F mutants had a 2-4-fold decreased affinity to fractalkine compared with wild type CX(3)CR1. By surface plasmon resonance measurements of fractalkine binding to biosensor chip-immobilized cell membranes, CX(3)CR1-Y14F mutants had a 100-fold decreased affinity to fractalkine. CX(3)CR1-expressing cell membranes treated with arylsulfatase to desulfate tyrosine residues also showed a 100-fold decreased affinity for fractalkine. Finally, synthesized, sulfated N-terminal CX(3)CR1 peptides immobilized on biosensor chips showed a higher affinity for fractalkine than non-sulfated peptides. Thus, we conclude that sulfation of tyrosine 14 enhances the function of CX(3)CR1 in cell capture and firm adhesion. Further, tyrosine sulfation may represent a general mechanism utilized by molecules that function in the rapid capture of circulating leukocytes.
Fong
Alan M
AM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Alam
S Munir
SM
Imai
Toshio
T
Haribabu
Bodduluri
B
Patel
Dhavalkumar D
DD
D000595:Amino Acid Sequence; D000073883:CX3C Chemokine Receptor 1; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D018121:Receptors, Cytokine / Q000378:metabolism*; D015728:Receptors, HIV / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D000073883:CX3C Chemokine Receptor 1; C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; D018076:DNA, Complementary; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018121:Receptors, Cytokine; D015728:Receptors, HIV; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D010649:Phenylalanine