Calcium requires cAMP to induce acrosomal exocytosis through a PKA-independent, Epac-mediated pathway

BRANHAM MT, MAYORGA L Y TOMES C

Pucón, Chile

Simposio; 13th International Symposium of Chromaffin Cell Biology; 2006

ISCCB (International Symposium on Chromaffin Cell Biology)

The acrosome reaction (AR) is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm (1). Calcium is the paradigmatic trigger of regulated exocytosis and we show here that its effects on acrosomal release are fully mediated by the second messenger cyclic adenosine 3´, 5´-monophosphate (cAMP). In sperm, various signaling pathways required to achieve egg-fertilizing ability depend on the intracellular rise of cAMP.  Most of these processes were thought to be mediated by cAMP-dependent protein kinases (PKA). Here we report a new pathway for cAMP-induced AR involving Epac.  Epac is a Rap-specific guanine-nucleotide exchange factor which is activated by the binding of cAMP to a cyclic nucleotide monophosphate-binding domain (2;3).  Calcium failed to trigger the AR when intracellular cAMP was depleted by an excess phosphodiesterase or when Epac was sequestered by specific blocking antibodies.  The non-discriminating dibutyrylcAMP and the Epac-selective 8-pCPT-2Me-cAMP analogues triggered the AR in the absence of cytosolic calcium.  This indicates that cAMP - via Epac activation - has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including Rab3A- induced tethering and SNARE-mediated docking of the acrosome to the plasma membrane, priming of the fusion machinery by NSF/a-SNAP, mobilization of intravesicular calcium through IP3-responsive channels, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-a-SNAP, anti-NSF and anti-Rab3A antibodies, to intra-acrosomal calcium chelators, to a calcium pump and channel blockers, and to botulinum toxins, but was resistant to PKA blockers.  These experiments now identify Epac in human sperm and define its indispensable role in exocytosis.