EPAC ACTIVATES THE SMALL G PROTEINS Rap1 AND Rab3A TO ACHIEVE

BRANHAM MARIA T; BUSTOS MATIAS A; DE BLAS GERARDO A; REHMANN HOLGER; ZARELLI VALERIA EP; TREVIÑO CLAUDIA L; DARSZON ALBERTO; MAYORGA LUIS S; TOMES CLAUDIA N

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2009 vol. 284 p. 24825 - 24839

0021-9258

Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium and mobilization from IP3-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in an A-kinase- and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac selective cAMP analogue 8-pCPT-2´-O-Me-cAMP induces a phospholipase C-dependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of sAC/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2´-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place, and another that elicits intracellular calcium release.