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 Cdependent 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.

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

“…We previously reported that sperm contain a GEF activity that exchanges GDP for GTP on Rab3A when challenged with calcium or 8-pCPT-2′-O-Me-cAMP (35,47). We conducted those earlier experiments in SLO-permeabilized human sperm, but because we performed the Rab27 pull-down experiments reported in this paper with nonpermeabilized sperm, we wished to verify the Rab3A data under the same conditions.…”

“…Because Rab27A and Rab27B have very similar electrophoretic mobility, and because the anti-Rab27 antibody that we used is supposed to recognize both isoforms, we cannot distinguish which of the two is present in sperm. By introducing specific anti-Rab antibodies that sequester the endogenous proteins or recombinant cassettes that selectively bind the active forms into SLO-permeabilized human sperm, we found that both Rab3A (32,35) (Fig. 4 and Fig.…”

“…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

“…As stated previously, acrosomal exocytosis is organized as a bifurcated pathway, with two separate limbs that diverge downstream of cAMP/Epac (28). The end point of one branch is the mobilization of intracellular calcium from internal stores through the activation of PLC via Rap, whereas the other branch (Rab3A-SNAREs) assembles the fusion protein machinery so that the outer acrosomal and plasma membranes become physically connected (protein branch).…”

“…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.…”