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.
Norgauer
J
J
Department of Dermatology, University of Freiburg, Germany.
Metzner
B
B
Czech
W
W
Schraufstatter
I
I
D000199:Actins / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D054360:Chemokine CXCL1; D018925:Chemokines / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000633:toxicity
D000199:Actins; D015703:Antigens, CD; C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D018076:DNA, Complementary; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D009363:Neoplasm Proteins; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D010738:Type C Phospholipases