Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis

María T Branham,Matías A Bustos,Gerardo A De Blas,Holger Rehmann,Valeria E.P Zarelli,Claudia L Treviño,Alberto Darszon,Luis S Mayorga,Claudia N Tomes

doi:10.1074/jbc.m109.015362

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.

Highlights

In certain neurons, neuroendocrine and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis
SAC Synthesizes cAMP Required for Human Sperm Exocytosis— Previous findings from our laboratory [8, 49] had led us to formulate the AR as summarized in Pathway 1, where single arrows indicate there is one step between the terms connected, and double arrows indicate that the number of steps taking place between the connected terms is unknown
For Rap proteins; we asked if Rap is present in human sperm and if its activation is required for exocytosis

Summary

Introduction

Neuroendocrine and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis. The sole addition of adenophostin rescued exocytosis impaired by anti-Rap1 antibodies, Ral-GDS-RBD, and U73122 (Fig. 3C), supporting the notion that the end point of the Rap-PLC pathway is the mobilization of intracellular calcium.

Results

Conclusion