Functional adhesiveness of the CX3CL1 chemokine requires its aggregation. Role of the transmembrane domain.
In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.
Hermand
Patricia
P
Laboratoire d'Immunologie Cellulaire, INSERM UMR-S 543, Université Pierre et Marie Curie-Paris 06, 91 boulevard de l'Hôpital, 75013 Paris, France.
Pincet
Frédéric
F
Carvalho
Stéphanie
S
Ansanay
Hervé
H
Trinquet
Eric
E
Daoudi
Mehdi
M
Combadière
Christophe
C
Deterre
Philippe
P
D000595:Amino Acid Sequence; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002448:Cell Adhesion; D002460:Cell Line; D054428:Chemokine CX3CL1 / Q000378:metabolism*; D017353:Gene Deletion; D006031:Glycosylation; D006801:Humans; D007249:Inflammation; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D000893:Anti-Inflammatory Agents; D054428:Chemokine CX3CL1