Disruption of the actin cytoskeleton with latrunculin A controls ATP-induced nadph oxidase activity and the recruitment of G proteins by neutrophil Gαq-coupled receptors

Abstract

Signaling by FPR1, the prototype G protein-coupled receptor (GPCR) expressed in neutrophils, is initiated by an activation of a G protein containing a Gα_i subunit. FPR1 activation results in an increase in the cytosolic concentration of free calcium ions ([Ca²⁺]_i), and an activation of the NADPH oxidase. Signals generated by the ATP receptor P2Y₂ are transduced by a Gα_q containing G protein. The neutrophil response induced by ATP includes a transient rise in [Ca²⁺]_i, but not any activation of the NADPH oxidase. ATP can, however, activate this oxidase through a receptor transactivation mechanism dependent both on P2Y₂R and on the allosterically modulated free fatty acid receptor FFA2R. The signals whereby FFA2R is activated by the Gα_q-coupled ATP receptor operate from the cytosolic side of the plasma membrane. Furthermore, in neutrophils with a disrupted actin cytoskeleton, ATP (as well as platelet activating factor; recognized by the Gα_q-coupled PAFR) potently activates the NADPH oxidase. At high concentrations of the actin cytoskeleton disrupting drug latrunculin A the activation was partly reduced by Gα_q inhibition as well as by the Gα_i inhibitor pertussis toxin. The effects on the ATP-induced NADPH oxidase activity, of the Gα_q inhibitor and pertussis toxin were more and less pronounced, respectively, when the concentration of latrunculin A was reduced. Taken together, we show that the neutrophil actin cytoskeleton is part of the regulatory machinery that determines the activation of the NADPH oxidase and the G protein recruitment profile downstream of activated of two Gα_q-coupled GPCRs expressed in primary neutrophils.