title: Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis.
abstract: 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 inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A- 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 soluble adenylyl cyclase/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.
authors: Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología-Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina.
authors: María T
authors: MT
authors: Branham
authors: Matías A
authors: MA
authors: Bustos
authors: Gerardo A
authors: GA
authors: De Blas
authors: Holger
authors: H
authors: Rehmann
authors: Valeria E P
authors: VE
authors: Zarelli
authors: Claudia L
authors: CL
authors: Treviño
authors: Alberto
authors: A
authors: Darszon
authors: Luis S
authors: LS
authors: Mayorga
authors: Claudia N
authors: CN
authors: Tomes
mesh_terms: D020101:Acrosome Reaction; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D005306:Fertilization; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008297:Male; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000089804:Shelterin Complex; D013094:Spermatozoa / Q000378:metabolism; D034501:Telomere-Binding Proteins / Q000378:metabolism*
chemical_list: C437704:APOA5 protein, human; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D011994:Recombinant Proteins; D000089804:Shelterin Complex; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D002118:Calcium