HIGH-AFFINITY AND LOW-AFFINITY BINDING OF GRO-ALPHA AND NEUTROPHIL-ACTIVATING PEPTIDE-2 TO INTERLEUKIN-8 RECEPTORS ON HUMAN NEUTROPHILS

GROalpha 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, GROalpha and NAP-2 were labeled with I-125 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 K(d) value of 0.3 and 0.7 nM for GROalpha and NAP-2, respectively, and 55-70% of low affinity (K(d) = 30 nM). Two proteins of almost-equal-to 70 kDa and 44 kDa (p70 and p44) were specifically cross-linked with labeled GROalpha, NAP-2, and IL-8. Unlabeled IL-8 fully inhibited this cross-linking and the binding of labeled GROalpha 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 GROalpha and NAP-2 with low affinity (K(d) = 30 nM) and IL-8 with high affinity (K(d) = 0.4 nM). Exposure of neutrophil membranes to 100 muM guanosine 5'-[gamma-thio]triphosphate led to a 75-fold increase of the K(d) in almost-equal-to 60% of the IL-8 receptors. High-affinity receptors for GROalpha 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 GROalpha, NAP-2, and IL-8 (p70 and p44) that may differ in their mode of interaction with GTP regulatory proteins.