Reconstitution of chemokine-induced actin polymerization in undifferentiated human leukemia cells (HL-60) by heterologous expression of interleukin-8 receptors.

The chemokines interleukin-8 (IL-8) and GRO alpha bind in neutrophils to the interleukin-8 receptor alpha and beta (IL-8R alpha and beta) triggering reorganization of the actin cytoskeleton and activation of phospholipase C (PLC). Reconstitution of chemokine-induced activation of PLC indicated coupling of IL-8R alpha and beta to pertussis toxin-insensitive G alpha 16-proteins as well as to pertussis toxin-sensitive G alpha i2- or G alpha i3-proteins. To identify the signal transduction mechanisms of chemokine-induced actin response, undifferentiated human leukemia cells (HL-60 cells) constitutively expressing G alpha 16-, G alpha i2- and G alpha i3-proteins were chosen for reconstitution studies. Expression of recombinant receptors after transfection of the cells with the cDNA of IL-8R alpha and beta was confirmed by binding studies with radiolabeled ligands. IL-8R alpha bound IL-8 with high affinity (Kd approximately 1 nM) and GRO alpha with low affinity (Kd approximately 1 microM), whereas IL-8R beta bound both IL-8 and GRO alpha with high affinity (Kd approximately 1nM). Flow cytometric actin measurements indicated that high affinity ligand-receptor interactions in both receptor transfectants displayed inducible responses. Pretreatment of transfectants with pertussis toxin caused ADP-ribosylation of G-proteins and blocked chemokine-induced polymerization, indicating involvement of G alpha i2- or G alpha i3-proteins, but not G alpha 16-proteins in this response.