Abstract
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.
Highlights
In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and involved, along with its unique receptor CX3CR1, in various inflammatory processes
We know that the potent adhesiveness of CX3CR1 under flow requires that CX3CL1-chemokine domain (CD) have a high affinity for it [19], nothing is known about the quaternary structure of the ligand
bioluminescence resonance energy transfer (BRET) of CX3CL1 in the HEK Cell Line—To investigate by BRET the aggregation state of the chemokine CX3CL1 in its native membranous form, we developed various constructs of CX3CL1 chimera that expressed Luc or yellow fluorescence protein (YFP) on the cytoplasmic C-terminal side
Summary
In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and involved, along with its unique receptor CX3CR1, in various inflammatory processes. BRET of CX3CL1 in the HEK Cell Line—To investigate by BRET the aggregation state of the chemokine CX3CL1 in its native membranous form, we developed various constructs of CX3CL1 chimera that expressed Luc or YFP on the cytoplasmic C-terminal side.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
