Functional linkage of the Gro beta and IL-8 receptors on the surface of human neutrophils.
Gro beta and IL-8 are two pro-inflammatory cytokines with chemotactic activities on neutrophils. Binding studies were performed to ascertain whether their similar biological activities are mediated through the same receptor. Since Gro beta lacks tyrosine residues, recombinant Gro beta containing an additional carboxyterminal tyrosine residue (Gro beta-Tyr) was produced in transfected COS cells, purified to homogeneity and radiolabelled with 125INa. Saturation experiments using [125I]-Gro beta-Tyr allowed us to identify high affinity receptors on human neutrophils (Kd: 2 +/- 0.5 nM and Bmax: 4760 +/- 761 sites/cell). Experiments using [125I]-IL-8 as ligand, showed no significative differences in affinity (Kd: 4 +/- 0.9 nM) but about two times the number of sites (11316 +/- 1810 sites/cell). In competition experiments using [125I]-Gro beta-Tyr, unlabelled IL-8 and Gro beta-Tyr generated superposable displacement curves (IC50: 0.69 +/- 0.15 nM and 0.42 +/- 0.11 nM, respectively). Interesting, IL-8 binding sites could be down-regulated by Gro beta and IL-8, indicating that the two binding sites may be associated. Cross-linking experiments using [125I]-IL-8 revealed two major bands at 70 and 140 kDa, whereas experiments with [125I]-Gro beta-Tyr showed only the 70 kDa band. Taken together, these results suggest that the human neutrophil IL-8/Gro beta receptor is a dimeric complex with two high affinity binding sites for IL-8 and of those two, only one is shared by Gro beta.
Vita
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Unité Biochimie des Protéines, Sanofl Elf Biorecherches, Labège, France.
Lefort
S
S
Brouillaud
M J
MJ
Magazin
M
M
Guillemot
J C
JC
Ferrara
P
P
D001667:Binding, Competitive / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D003432:Cross-Linking Reagents; D015536:Down-Regulation / Q000502:physiology; D006801:Humans; D007457:Iodine Radioisotopes; D009504:Neutrophils / Q000737:chemistry*; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000737:chemistry*; D018123:Receptors, Interleukin / Q000737:chemistry*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B