Mutational analysis of the fractalkine chemokine domain. Basic amino acid residues differentially contribute to CX3CR1 binding, signaling, and cell adhesion.
Fractalkine (FKN/CX3CL1) is a unique member of the chemokine gene family and contains a chemokine domain (CD), a mucin-like stalk, a single transmembrane region, and a short intracellular C terminus. This structural distinction affords FKN the property of mediating capture and firm adhesion of FKN receptor (CX3CR1)-expressing cells under physiological flow conditions. Shed forms of FKN also exist, and these promote chemotaxis of CX3CR1-expressing leukocytes. The goal of the present study was to identify specific residues within the FKN-CD critical for FKN-CX3CR1 interactions. Two residues were identified in the FKN-CD, namely Lys-7 and Arg-47, that are important determinants in mediating an FKN-CX3CR1 interaction. FKN-K7A and FKN-R47A mutants exhibited 30-60-fold decreases in affinity for CX3CR1 and failed to arrest efficiently CX3CR1-expressing cells under physiological flow conditions. However, these mutants had differential effects on chemotaxis of CX3CR1-expressing cells. The FKN-K7A mutant acted as an equipotent partial agonist, whereas the FKN-R47A mutant had marked decreased potency and efficacy in measures of chemotactic activity. These data identify specific structural features of the FKN-CD that are important in interactions with CX3CR1 including steady state binding, signaling, and firm adhesion of CX3CR1-expressing cells.
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610-0267, USA.
J K
JK
Harrison
A M
AM
Fong
P A
PA
Swain
S
S
Chen
Y R
YR
Yu
M N
MN
Salafranca
W B
WB
Greenleaf
T
T
Imai
D D
DD
Patel
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000599:Amino Acids, Diamino; D000818:Animals; D001665:Binding Sites; D000073883:CX3C Chemokine Receptor 1; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000737:chemistry* / Q000502:physiology*; D002633:Chemotaxis / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D018121:Receptors, Cytokine / Q000378:metabolism*; D015728:Receptors, HIV / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D000599:Amino Acids, Diamino; D000073883:CX3C Chemokine Receptor 1; C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; C515539:Cx3cl1 protein, mouse; C515540:Cx3cl1 protein, rat; D008565:Membrane Proteins; D018121:Receptors, Cytokine; D015728:Receptors, HIV; D011994:Recombinant Proteins; D002118:Calcium