Characterization of interleukin-8 receptors in human neutrophil membranes: regulation by guanine nucleotides.

Interleukin-8 (IL-8) is a polymorphonuclear leukocyte (PMN) chemoattractant and activator which mediates its effects through specific cell-surface receptors. Indirect evidence indicates that guanine nucleotide regulatory proteins (G proteins) are necessary for transmembrane signaling. The present study characterizes IL-8 receptors in isolated PMN membrane fractions and shows direct regulation of these receptors by guanine nucleotides. The binding of [125I]IL-8 to subcellular fractions of PMNs showed specific binding in a low-density membrane fraction containing alkaline phosphatase, but not in primary or secondary granules. The binding of [125I]IL-8 was rapid and reversible. The equilibrium dissociation constant (Kd) of the receptor ranged from 5.0-12.4 nM and there were 1.58-5.90 . 10(10) receptors/mg protein. The dose-response curves for the competitive binding of three different forms of IL-8 to the receptor labeled by [125I]IL-8 corresponded with their ability to produce chemotaxis and granule exocytosis in PMNs. Treatment of membranes with the nonhydrolyzable analogs of GTP, GMP-PNP and GTP gamma S, inhibited the binding of [125I]IL-8. GMP-PNP decreased the affinity of the IL-8 receptor by approx. 2-fold without altering the total receptor number. These findings demonstrate that IL-8 receptors in PMN membranes are of high affinity and are convertible to a low-affinity state in the presence of guanine nucleotides, suggesting a direct role for G proteins in transmembrane signaling by this cytokine.