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J. Biol. Chem., Vol. 281, Issue 13, 8656-8666, March 31, 2006

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Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway^*

From the Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, Mendoza 5500, Argentina

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca²⁺ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca²⁺ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca²⁺. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca²⁺, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti--SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca²⁺ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca²⁺ in exocytosis.

Received for publication, August 11, 2005 , and in revised form, December 22, 2005.

^* This work was supported by an International Research Scholar Award from the Howard Hughes Medical Institute (to L. S. M.) and grants from Consejo Nacional de Investigaciones Científicas y Técnicas and Agencia Nacional de Promoción Científica y Tecnológica and from the Cuyo National University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1-S6.

¹ To whom correspondence should be addressed. Tel./Fax: 54-261-449-4143; E-mail: ctomes{at}fcm.uncu.edu.ar.

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