Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis.
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.
Branham
María T
MT
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.
Bustos
Matías A
MA
De Blas
Gerardo A
GA
Rehmann
Holger
H
Zarelli
Valeria E P
VE
Treviño
Claudia L
CL
Darszon
Alberto
A
Mayorga
Luis S
LS
Tomes
Claudia N
CN
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*
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