High- and low-affinity binding of GRO alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils.
GRO alpha and neutrophil-activating peptide 2 (NAP-2), like their analog interleukin 8 (IL-8), are considered to be inflammatory mediators since they recruit and activate neutrophil leukocytes. After introduction of tyrosines by substitution for other residues at the C terminus, GRO alpha and NAP-2 were labeled with 125I and used for binding studies. A total of 60,000-90,000 receptors per neutrophil were found for either ligand. Of these 30-45% were of high affinity with a mean Kd value of 0.3 and 0.7 nM for GRO alpha and NAP-2, respectively, and 55-70% of low affinity (Kd = 30 nM). Two proteins of approximately 70 kDa and 44 kDa (p70 and p44) were specifically cross-linked with labeled GRO alpha, NAP-2, and IL-8. Unlabeled IL-8 fully inhibited this cross-linking and the binding of labeled GRO alpha or NAP-2 to the high-affinity sites on neutrophils or neutrophil membranes. Treatment of membranes with digitonin resulted in the preferential solubilization of a single receptor species, corresponding to p44, that bound GRO alpha and NAP-2 with low affinity (Kd = 30 nM) and IL-8 with high affinity (Kd = 0.4 nM). Exposure of neutrophil membranes to 100 microM guanosine 5'-[gamma-thio]triphosphate led to a 75-fold increase of the Kd in approximately 60% of the IL-8 receptors. High-affinity receptors for GRO alpha and NAP-2 were similarly affected. In contrast, guanosine 5'-[gamma-thio]triphosphate had no effect on the binding of IL-8 to p44 solubilized by digitonin. These results demonstrate that human neutrophils bear two classes of receptors for GRO alpha, NAP-2, and IL-8 (p70 and p44) that may differ in their mode of interaction with GTP regulatory proteins.
Schumacher
C
C
Theodor-Kocher Institute, University of Bern, Switzerland.
Clark-Lewis
I
I
Baggiolini
M
M
Moser
B
B
D002462:Cell Membrane / Q000378:metabolism; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000378:metabolism*; D007700:Kinetics; D019272:Leukocyte Elastase; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D013329:Structure-Activity Relationship; D001620:beta-Thromboglobulin
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; C061615:PPBP protein, human; D010455:Peptides; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A; D001620:beta-Thromboglobulin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C009862:connective tissue-activating peptide; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase