Activation of the small GTPase rap1 in human neutrophils.
The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b558 or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca2+ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca2+ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca2+ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation.
M'Rabet
L
L
Laboratory for Physiological Chemistry and Department of Pulmonary Diseases, Utrecht University, Utrecht, The Netherlands; and the University of London Hospital, London, UK.
Coffer
P
P
Zwartkruis
F
F
Franke
B
B
Segal
A W
AW
Koenderman
L
L
Bos
J L
JL
D015232:Dinoprostone / Q000494:pharmacology; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010972:Platelet Activating Factor / Q000494:pharmacology; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins
D010972:Platelet Activating Factor; D020726:ral Guanine Nucleotide Exchange Factor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D015232:Dinoprostone