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

• Published in: Inflammation research 1996-03, Vol.45 (3), p.127-131
• Main Authors: Norgauer, J, Metzner, B, Czech, W, Schraufstatter, I
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Adenosine Diphosphate Ribose - chemistry; Antigens, CD - genetics; Antigens, CD - metabolism; Base Sequence; Binding, Competitive; Chemokine CXCL1; Chemokines - genetics; Chemokines - metabolism; Chemokines - pharmacology; Chemokines, CXC; Chemotactic Factors - genetics; Chemotactic Factors - metabolism; Chemotactic Factors - pharmacology; DNA, Complementary - chemistry; DNA, Complementary - metabolism; Enzyme Activation - drug effects; Growth Substances - genetics; Growth Substances - metabolism; Growth Substances - pharmacology; HL-60 Cells - cytology; HL-60 Cells - drug effects; HL-60 Cells - metabolism; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8 - genetics; Interleukin-8 - metabolism; Interleukin-8 - pharmacology; Molecular Sequence Data; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Neoplasm Proteins - pharmacology; Neutrophils - cytology; Neutrophils - drug effects; Neutrophils - metabolism; Pertussis Toxin; Polymerase Chain Reaction; Receptors, Interleukin - genetics; Receptors, Interleukin - metabolism; Receptors, Interleukin-8A; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Signal Transduction - drug effects; Transfection; Type C Phospholipases - metabolism; Virulence Factors, Bordetella - toxicity
• ISSN: 1023-3830; 1420-908X
• DOI: 10.1007/bf02265165

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.