Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis

BRANHAM, M.T; BUSTOS, MA; DE BLAS, G.A.; REHMANN, H.; ZARELLI, V.E.; TREVIÑO, C.L.; DARSZON, A.; MAYORGA, LS; TOMES, C.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 acrosomereaction) relies on cAMP production, assembly ofa proteinaceous fusion machinery, calcium influxfrom the extracellular medium and mobilizationfrom IP3-sensitive intracellular stores. Additionof cAMP to human sperm suspensions bypassessome of these requirements and elicits exocytosisin an A-kinase- and extracellularcalcium-independent manner. The relevantcAMP target is Epac, a guanine nucleotideexchange factor for the small GTPase Rap. Weshow here that a soluble adenylyl cyclasesynthesizes the cAMP required for the acrosomereaction. Epac stimulates the exchange of GDPfor GTP on Rap1, upstream of a phospholipaseC. The Epac selective cAMP analogue8-pCPT-2´-O-Me-cAMP induces a phospholipaseC-dependent calcium mobilization in humansperm suspensions. In addition, our studiesidentify a novel connection between cAMP andRab3A, a secretory granule-associated protein,revealing that the latter functions downstream ofsAC/cAMP/Epac but not of Rap1. Challengingsperm with calcium or 8-pCPT-2´-O-Me-cAMPboosts the exchange of GDP for GTP on Rab3A.Recombinant Epac does not release GDP fromRab3A in vitro, suggesting that the Rab3A-GEFactivation by cAMP/Epac in vivo is indirect. Wepropose that Epac sits at a critical point duringthe exocytotic cascade after which the pathwaysplits into two limbs, one that assembles thefusion machinery into place, and another thatelicits intracellular calcium release.