RhoG Regulates the Neutrophil NADPH Oxidase

Abstract

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG−/−) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG−/− neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG−/− neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.

P-Rex1 directly activates RhoG to regulate GPCR-driven Rac signalling and actin polarity in neutrophils

“…The striking defect we observed in DOCK2 recruitment to the leading edge of polarised neutrophils upon loss of RhoG indicates that this mechanism is also responsible for linking RhoG and Rac downstream of GPCRs in neutrophils. It is also consistent with previous data indicating P-Rex 2/2 (Dong et al, 2005;Welch et al, 2005), RhoG 2/2 (Condliffe et al, 2006) and DOCK2 2/2 (Kunisaki et al, 2006) neutrophils all display defects in GPCRinduced Rac activity and ROS production. This new pathway for GPCR-mediated control of Rac thus unifies a number of confusing observations in the field of Rho family signalling, by indicating a hierarchical, rather than simply cooperative organisation of GEF input into Rac activation (Fig.…”

“…A key question, therefore, is the extent to which activation of RhoG is the most important function of P-Rex1 and, additionally, the relationship of this function to the activation of its alternative potential substrates Rac1 and Rac2. Separate studies have previously reported that RhoG and P-Rex1 regulate some Rac2-dependent neutrophil functions, such as activation of the NADPH oxidase (Condliffe et al, 2006;Dong et al, 2005;Welch et al, 2005). However, the relatively small effects of P-Rex1 deletion observed in these studies contrast with the more severe defects reported upon RhoG deletion, and thus conflict with our observation that P-Rex1 is the major regulator of RhoG in this system.…”

“…There is evidence for a role of RhoG downstream of GPCR engagement in neutrophils (Condliffe et al, 2006); however, its activation and regulation has yet to be measured in any GPCRdriven system. In order to assess the regulation of RhoG downstream of GPCRs, we developed a refined procedure for purifying primary mouse neutrophils in a quiescent state that allows them to respond robustly and rapidly to GPCR ligands, such as the formylated peptide N-formyl-methionyl-leucylphenylalanine (fMLP), with minimal pre-activation by autocrine or paracrine factors (see Materials and Methods).…”

“…Instead, it has been reported to function upstream of Rac in some systems by recruiting a bipartite Rac GEF complex comprising members of the DOCK (isoforms 1-5) and ELMO (isoforms 1-3) families (Côté et al, 2002;Katoh and Negishi, 2003). In addition to roles in EGF-dependent cell migration, neurite outgrowth, endocytosis and leukocyte transendothelial migration, RhoG is required for efficient activation of the NADPH oxidase by GPCR ligands in mouse neutrophils (Bass et al, 2011;Condliffe et al, 2006;Hiramoto-Yamaki et al, 2010;Samson et al, 2010;van Buul et al, 2007). Furthermore, mouse neutrophils lacking DOCK2, the major DOCK protein in neutrophils, have severe defects in GPCR-stimulated ROS production and cell migration (Kunisaki et al, 2006).…”

Yersinia enterocolitica differentially modulates RhoG activity in host cells

“…It has been established that binding of active RhoG to a complex of Elmo and Dock180 stimulates the GEF activity of Dock180, leading to GTP-loading of Rac1 (deBakker et al, 2004;Katoh and Negishi, 2003;Lu and Ravichandran, 2006). Interestingly, RhoG has been implicated in various cell functions known to be subdued by Yersinia, such as integrin-mediated phagocytosis, transendothelial migration and activation of NADPH oxidase (Condliffe et al, 2006;Nakaya et al, 2006;van Buul et al, 2007).…”