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  • Epac proteins consist of a carboxyl terminal


    Epac proteins consist of a carboxyl-terminal catalytic region and an amino-terminal regulatory region, which harbors one cAMP-binding domain in Epac1 and two in Epac2 [22], [32], [51]. In the absence of cAMP, the regulatory region covers the CDC25-homology domain and autoinhibits Epac\'s enzymatic activity by hindering the access of its substrates to the catalytic site [6], [7], [45]. Upon binding of cAMP, a subtle conformational change allows the regulatory region to move away, lifting the autoinhibition [5], [22]. Epac\'s catalytic portion bears a GEF activity specific for Rap1 and Rap2 [17], [29]. The small GTPase Rap1 is necessary to achieve acrosomal exocytosis [8], [35], [47]. Importantly, Rap1 exchanges GDP for GTP in response to calcium and 8-pCPT-2′-O-Me-cAMP [2], [8], [38], [47]. One of the advances reported here is that progesterone, perhaps the most widely used AR inducer, increases the population of Lonidamine with active Rap1 in the acrosomal region. Equally important is the finding that progesterone requires cAMP but not PKA to activate Rap1. In many models, cAMP/PKA and/or cAMP/Epac facilitate the opening of calcium release channels located in intracellular stores. Metabolites such as inositol 1,4,5-trisphosphate (IP3), cyclic ADP-ribose (the proposed endogenous ligand for ryanodine receptors), and NAADP enhance the ability of cytosolic calcium to activate various calcium release channels located on intracellular organelles [30], [32]. Pharmacological blocking of these pathways impairs glucose-induced insulin secretion, indicating that intracellular calcium is required for exocytosis [39]. What could be the link between cAMP/Epac and intracellular calcium mobilization in secretory cells? An interesting candidate is PLCε, the only effector recruited/activated by GTP-bound Rap that has been implicated in Epac-mediated secretory responses [18], [19]. In sperm, the acrosome itself behaves as an internal calcium store [14], [16], [43], [47], [49]. We have previously shown by single-cell confocal microscopy that intra-acrosomal calcium is released through IP3-sensitive channels because the blocker 2-aminoethoxydiphenylborate (2-APB) prevents – and the agonist adenophostin A elicits – its mobilization [47]. The sensitivity of the AR to these agents provides pharmacological evidence that intracellular calcium is mobilized via IP3-sensitive channels in human sperm [8], [16], [35]. One of the significant advances that we describe in this manuscript is that PLCε is required to mobilize calcium from the acrosomal reservoir. The stimulation of sperm\'s Epac with 8-pCPT-2′-O-Me-cAMP suffices to elicit intra-acrosomal calcium efflux through a pathway that involves Rap1 and PLCε.
    Materials and methods
    Discussion An influx of calcium into the cytosol through channels in the plasma membrane almost universally triggers the fusion of secretory vesicles with the cell membrane. Frequently, calcium mobilized from internal sources is also necessary for exocytosis (see Section 1). The sperm model is ideally suited to address the question of how does cAMP connect to calcium mobilization because (i) exocytosis can proceed in the absence of external calcium (which eliminates potentially confounding recordings), (ii) the acrosome itself is a reservoir of releasable calcium, and (iii) sperm do not undergo any trafficking processes other than exocytosis.