MIF-chemokine receptor interactions in atherogenesis are dependent on an N-loop-based 2-site binding mechanism.
Macrophage migration inhibitory factor (MIF) is a cytokine that mediates inflammatory diseases. MIF promotes atherogenic leukocyte recruitment through a promiscuous, yet highly affine, interaction with CXCR2 and CXCR4. Binding to CXCR2 is dependent on a pseudo-(E)LR motif in MIF, but a second interaction site has been elusive. Here we identified an N-like loop in MIF, suggesting that MIF binding to CXCR2 follows the 2-site binding mode of bona fide chemokines. For MIF, the model predicts interactions between the N-like loop and the CXCR2 N domain (site 1) and pseudo-(E)LR and extracellular loops (ELs) of CXCR2 (site 2). Applying biophysical and peptide array analysis, we demonstrated an interaction between MIF and the CXCR2 N domain, which was pseudo-(E)LR independent. Peptide array analysis also indicated that the pseudo-(E)LR motif is responsible for MIF binding to EL2 and 3. Notably, peptides MIF-(40-49) and MIF-(47-56), representing N-like-loop-derived peptides, but not a scrambled control peptide, significantly blocked MIF/CXCR2 binding, MIF-mediated monocyte arrest under flow on aortic endothelial cells in vitro (IC(50): 1.24×10(-6) M), and MIF-dependent monocyte adhesion to atherosclerotic mouse carotid arteries in vivo. Thus, the N-like loop in MIF is critical for MIF's noncognate interaction with CXCR2 and proatherogenic functions. The 2-site binding model that explains chemokine receptor activation also applies to MIF.
Institute of Biochemistry and Molecular Cell Biology, Rheinisch-Westfälische Technische Hochschule Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Sandra
S
Kraemer
Hongqi
H
Lue
Alma
A
Zernecke
Aphrodite
A
Kapurniotu
Erika
E
Andreetto
Ronald
R
Frank
Birgitt
B
Lennartz
Christian
C
Weber
Jürgen
J
Bernhagen
D000818:Animals; D001011:Aorta / Q000166:cytology; D001057:Apolipoproteins E / Q000235:genetics; D050197:Atherosclerosis / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D002942:Circular Dichroism; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009000:Monocytes / Q000166:cytology; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D023063:Receptors, Interleukin-8B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001057:Apolipoproteins E; C508606:CXCL8 protein, human; D016209:Interleukin-8; D008263:Macrophage Migration-Inhibitory Factors; D011971:Receptors, Immunologic; D023063:Receptors, Interleukin-8B; C033184:macrophage migration inhibitory factor receptor; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human; C076231:Mif protein, mouse