Afuresertib receptor Sperm exocytosis or acrosome reaction A

Sperm exocytosis or acrosome reaction (AR) is a regulated secretion with special characteristics essential for fertilization. The AR relies on the same highly conserved molecules that drive intracellular membrane fusion and exocytosis in all other Afuresertib receptor (see [59] for a recent review). Two such molecules are Rabs27 and 3; they are predominantly in their inactive conformation in resting sperm and exchange GDP for GTP in response to exocytotic stimuli [9,11,43]. Rab27 acts in concert with, and plays its role upstream of, Rab3 [9,11]. Rab27A-GTP recruits a Rab3AGEF from human sperm extracts [9]. The Rab27 → Rab3GEF → Rab3 sequence remains the only RabGEF cascade described in dense-core vesicle exocytosis in mammals to date.
Materials and methods
Results
Discussion The identification of the Rab3GEF has been underscored by controversy. In 1994, Mss4 was characterized as the first GDP releasing factor for Rab3 [8]. Subsequently, three other groups measured an exchange activity of Mss4 toward Rab3 [17,63,68]. It was later shown that Mss4 is promiscuous in binding exocytotic Rabs, including Rab3A, and that it exhibits a weak exchange activity toward this isoform [65]. An alternative role has been proposed for Mss4: that of a chaperone that stabilizes Rabs other than Rab3 in GLUT4 exocytosis; Mss4's GEF activity is dispensable for this function [32]. The second protein reported to have Rab3GEF activity was purified, cloned, sequenced and named Rab3GEP. Unlike Mss4, Rab3GEP is specific for lipid-modified Rab3s, which correspond to the endogenous isoforms [63]. Later on, Rab3GEP's GEF activity toward bacterially expressed, non geranylgeranylated Rab3A was detected by [24] but not by [67]. Rab3GEP was classified as belonging to the MADD (MAPK-activating protein containing a death domain)/DENN (differentially expressed in normal and neoplastic cells) family [17,45,48]. It has been proposed that Rab3GEP is a Rab3-GTP effector essential for the axonal transport of Rab3 rather than a Rab3GEF [51]. Furthermore, Rab3GEP/MADD, has been reported to exhibit GEF activity in vitro toward Rab27A and B expressed in Escherichia coli and non-lipid modified [24,67] and in vivo toward endogenous Rab27 in pancreatic acinar cells [36]. The third protein described as a Rab3GEF both in vitro and in vivo [24,44] is GRAB. Yoshimura and collaborators also measured exchange activity toward Rab3, albeit much weaker that toward Rab8 [67], whereas another group detected GRAB's exchange activity toward Rab8 but not Rab3 [33]. It is worth pointing out that we offered geranygeranylated Rab3A as substrate, which might explain why we readily detected GRAB's catalytic activity (Fig. 2E). The amino-terminal portion of GRAB binds Rab3 whereas its carboxy-terminal domain binds Rab11. This dual Rab binding property has led to the speculation that GRAB could function in a Rab cascade in which its activity as an effector for one Rab (Rab11A/Rab11B) could be coupled to its activity as a GEF to another (Rab3A/Rab8A/Rab8B) [35]. The cascade Rab11 → GRAB→Rab8 has recently been shown to govern axonal growth [30]. A Rab11 → Rabin8 → Rab8 cascade is required for primary cilium [40] and apical membrane [7] formation (reviewed in [49]). Unlike the ones mentioned before, the cascade we propose here (Rab27→Rabphilin3a→GRAB→Rab3) consists of two Rabs and a GEF plus an effector for the first Rab. Although some of our data would be consistent with a simpler Rab27→GRAB→Rab3 cascade, we do not favor this view because Rabphilin3a is necessary for the activation of Rab3 (Fig. 7B). Rab27 has been proposed to dock exocytotic granules to the plasma membrane through its interaction with effectors, which, in turn, bind the SNARE protein SNAP25, SNARE-interacting proteins of the Munc18 family and/or phospholipids (see examples in Fig. 2 in [26]). In contrast, it would appear that more than one or two molecules separate Rab27 from the plasma membrane in the sperm model (Fig. 5 in [11]).