Critical role of Ral GTPase as a gatekeeper in controlling neutrophil selective degranulation of secondary granules (140.11)

Abstract

Abstract Neutrophil (PMN) inflammatory responses, including cell adhesion, diapedesis and phagocytosis, are dependent on the mobilization and release of various intracellular granules and vesicles. In this study, we observed that inhibition of the Ras family GTPases by damnacanthal and manumycin A resulted in specifically release of PMN secondary granules, but not primary or tertiary granules, in the absence of stimulation. By screening Ras family members for activity changes during PMN activation and degranulation, we identified Ral GTPase to be a critical regulator that selectively controls secondary granule release. In particular, we found that Ral exists as an active GTPase in resting PMN, while quick inactivation correlates with chemoattractant-induced degranulation. Indeed, we demonstrated that over-expression of a constitutively active Ral mutant (Ral23V) in PMN completely blocked secondary granule release. Subcellular fractionation and immunofluorescence labeling further found that Ral is associated with the plasma membrane and redistributed to secondary granules following chemoattractant stimulation. Blocking clathrin-mediated plasma membrane endocytosis, however, inhibited Ral intracellular translocation and PMN degranulation. Thus, these results provide strong evidence demonstrating Ral to be an essential gatekeeper for secondary granules in PMN. Dysregulation of Ral results in impairment of PMN transmigration in response to inflammatory stimuli.