Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

Abstract

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/ Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fc␥ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fc␥ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (Fc␥R) 4 mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of Fc␥R on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin-and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, crosslinking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, Fc␥R possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho-and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase C␥ (PLC␥), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for Fc␥R-induced phagocytosis (7,8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidyli...

Ral Isoforms Are Implicated in FcγR-Mediated Phagocytosis: Activation of Phospholipase D by RalA

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Phagocytosis: A Fundamental Process in Immunity

“…These receptors initiate intracellular signaling that activates the small GTPase Rap1 [66], which in turn provokes conformational changes in the integrin, leading to its increased affinity. This process is called inside-out signaling because the signal that activates the integrin comes from inside the cell.…”

RasGRP Ras guanine nucleotide exchange factors in cancer

“…Within the hematopoietic system, RasGRP2's expression is restricted to platelets and their precursors, megakaryocytes as well as neutrophils (Crittenden et al, 2004; Cifuni et al, 2008; Carbo et al, 2010). RasGRP3 is highly expressed in B cells (Yamashita et al, 2000; Teixeira et al, 2003) but is also expressed in T cells, macrophages and endothelial cells (Yamashita et al, 2000; Teixeira et al, 2003; Roberts et al, 2004; Botelho et al, 2009). Finally, RasGRP4 is the only family member, which is does not appear to be expressed in the brain (Reuther et al, 2002).…”