Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.

Abstract

Activation of human neutrophils involves the degranulation of specific and azurophil granules. This process is GTP-dependent and the presence of small GTP-binding proteins (SGBPs) has been detected in the two granule populations. At present, none of these SGBPs has been definitely identified. In order to characterize some of these proteins and obtain further insights as to their potential role in degranulation processes, we have used specific antibodies directed against the ras-related rap1 and rap2 proteins. By immunoblot analysis, we observed that rap2p is predominantly located in specific granules, whereas rap1p is detected both in specific granules and a fraction enriched in plasma membranes. Neither rap1p nor rap2p was found in the cytosol or in azurophil granules. Similarly, by indirect immunofluorescence, we observed that cytoplasmic granules were stained with anti-rap1p antibodies and anti-rap2p antibodies, and the plasma membrane was labeled with both antibodies but more distinctly with anti-rap1p than with anti-rap2p antibodies. rap1p and rap2p are tightly bound to the membrane of specific granules since they cannot be extracted by high salt or alkaline buffers. In addition, treatment of intact specific granules with pronase induced the degradation of rap proteins suggesting that they are exposed to the cytoplasmic face of the granules. Degranulation of neutrophils consists of the translocation and subsequent fusion of granules with the plasma membrane. Activation of this process induced the accumulation of rap proteins in the plasma membrane as observed by subcellular fractionation and indirect immunofluorescence experiments; this was not associated with the appearance of a soluble form of these proteins, showing that they remain membrane-bound during this process. The identification and subcellular localization of rap1p and rap2p at the surface of specific granules and the observation that they translocate to the plasma membrane upon cell stimulation without appearance of soluble forms constitute an important step toward the understanding of their physiological functions in human neutrophils.