Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway

Abstract

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca 2؉ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca 2؉ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyrylcAMP and the Epac-selective 8-(p-chlorophenylthio)-2-O-methyladenosine-3,5-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca 2؉ . This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca 2؉ , and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-␣-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca 2؉ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca 2؉ in exocytosis.

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis

“…The acrosome behaves as an internal store of releasable calcium (41)(42)(43)(44); efflux from this reservoir through inositol 1,4,5 triphosphate (IP 3 )-sensitive channels is required for the AR initiated by all inducers (32,33,41,45). We had previously demonstrated that Rab3A acts in the AR before the release of calcium from the intracellular store (32).…”

“…Here we focus on the role of two secretory Rabs, Rab3A and Rab27, in the release of the sperm single dense-core secretory granule. Over the last several years, we have published several papers that demonstrate the positive role of Rab3A in sperm exocytosis (32,33); in this paper, we describe the presence of Rab27 in sperm and its role in the AR. Rab3A and Rab27A are phylogenetically similar (54); Rab27A and Rab27B have only moderate homology to other human Rab sequences, with the greatest homology (41-44%) to members of the Rab3 subfamily (40).…”

“…Rab3A is present in the acrosomal region of human (28), rat, and mouse sperm (29); in the latter it is predominantly membrane-bound and disappears from cells that have undergone the AR (30). Rab3A is required for the AR triggered by calcium (31,32), cAMP (33), and sphingosine 1-phosphate (34) in human sperm. Treatment with AR inducers increases the levels of GTP-bound Rab3A (35).…”

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

“…During exocytosis, there are some parallel and concomitant steps necessary to achieve AR that occurs thereafter. This signaling cascade involves preparation of the fusion machinery that includes SNAREs (21,22), ␣-SNAP/NSF (23), synaptotagmin (24), Rab3A (25,26), and exchange protein directly activated by cAMP (Epac), a GEF protein that links cAMP and intracellular calcium signaling (27,28). It has been proposed that a soluble adenylyl cyclase is activated by a sustained increase in intracellular calcium, resulting in a cAMP increase that activates Epac.…”

The Epac-Rap1 Signaling Pathway Controls cAMP-mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells

“…Several reports have documented signaling pathways independent of PKA that may be involved in the regulation of cAMP-mediated secretory vesicle release. More specifically, the exchange protein activated by cAMP (Epac) has been implicated in cAMP-mediated vesicle exocytosis (17,18). In this study, we explored a potential role for the cAMP-guanine nucleotide exchange factor Epac and its substrate Rap1 in the regulation of WPB exocytosis by human primary endothelial cells.…”