Abstract
BackgroundThe small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex.ResultsWe investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges.ConclusionsOur results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.
Highlights
Neutrophils are rapidly polarized upon the detection of a chemoattractant gradient, and start to migrate toward the chemoattractant source
Recruitment of Gβγ, αPIX, and p21-activating protein kinase 1 (PAK1) to leading edges occur independent of the GBF1 Silencing of GBF1 in HL-60 cells frequently generated multi-head leading edges during fMLP-induced chemotaxis, similar to those observed upon the inhibition of RAC1 or CDC42 [15]
We have shown that GBF1 small interfering RNA treatment causes loss of the polarized accumulation of GIT2 at the leading edges of fMLP-stimulated HL-60 cells, in which a greater than 50% decrease in the accumulation of GIT2 at actin-rich leading edges was observed compared with cells treated with a control irrelevant siRNA [15]
Summary
Neutrophils are rapidly polarized upon the detection of a chemoattractant gradient, and start to migrate toward the chemoattractant source Such directional cell migration requires a complex but well organized series of intracellular events, such as cytoskeleton remodeling, and membrane trafficking and remodeling. The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which employs PAK1. It has remained unclear as to why GIT2 is included in this complex
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