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• W2048096689 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. 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. During fertilization in eutherian mammals, the spermatozoon must penetrate the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR) 4The abbreviations used are:ARacrosome reaction2-APB2-aminoethoxydiphenyl borate6-Bnz-cAMPN6-benzoyladenosine-3′,5′-cyclic monophosphateCICRcalcium-induced calcium releaseEpacexchange protein directly activated by cAMPFITC-PSAfluorescein isothiocyanate-coupled Pisum sativum agglutininGEFguanine-nucleotide exchange factorGDIguanine nucleotide-dissociation inhibitormGDP2′,3′-O- (N′-methylanthraniloyl)-GDPNSFN-ethylmaleimide-sensitive factorNP-EGTA-AMO-nitrophenyl EGTA acetoxymethyl ester8-pCPT-2′-O-Me-cAMP8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphatePKAcAMP-dependent protein kinasePDEphosphodiesterasePLCphospholipase CPMSFphenylmethylsulfonyl fluoridePTP1Bprotein-tyrosine phosphatase 1BsACsoluble adenylyl cyclaseSLOstreptolysin OTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineIP3inositol 1,4,5-trisphosphatePBSphosphate-buffered salineGTP-γ-Sguanosine 5′-3-O-(thio)triphosphateGSTglutathione S-transferaseSNAREsoluble NSF attachment protein receptorsPI-PLCphosphatidylinositol-specific phospholipase C. can successfully accomplish this task (1Florman H.M. Ducibella Elsevier-T. Neill J.D. Knobil and Neill's Physiology of Reproduction. Elsevier Academic Press, San Diego, CA2005: 55-112Google Scholar). The AR is a regulated exocytosis where the membrane of the acrosome, the single dense core secretory granule in sperm, fuses to the plasma membrane surrounding the anterior portion of the head. This process releases hydrolytic enzymes stored in the granule. These enzymes, together with the physical thrust derived from strong flagellar beating, enable sperm to penetrate the zona pellucida (1Florman H.M. Ducibella Elsevier-T. Neill J.D. Knobil and Neill's Physiology of Reproduction. Elsevier Academic Press, San Diego, CA2005: 55-112Google Scholar, 2Bedford J.M. Biol. Reprod. 1998; 59: 1275-1287Crossref PubMed Scopus (126) Google Scholar). Physiological agonists accomplish the AR by inducing an influx of calcium from the extracellular medium and the assembly of a conserved proteinaceous fusion machinery that includes Rab3A, α-SNAP/NSF, synaptotagmin, complexin, and neurotoxin-sensitive SNAREs; the AR also requires an efflux of calcium from inside the acrosome through IP3-sensitive channels (reviewed in Refs. 3Mayorga L. Tomes C.N. Belmonte S.A. IUBMB Life. 2007; 59: 286-292Crossref PubMed Scopus (79) Google Scholar, 4Tomes C.N. Soc. Reprod. Fertil. Suppl. 2007; 65: 275-291PubMed Google Scholar). acrosome reaction 2-aminoethoxydiphenyl borate N6-benzoyladenosine-3′,5′-cyclic monophosphate calcium-induced calcium release exchange protein directly activated by cAMP fluorescein isothiocyanate-coupled Pisum sativum agglutinin guanine-nucleotide exchange factor guanine nucleotide-dissociation inhibitor 2′,3′-O- (N′-methylanthraniloyl)-GDP N-ethylmaleimide-sensitive factor O-nitrophenyl EGTA acetoxymethyl ester 8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate cAMP-dependent protein kinase phosphodiesterase phospholipase C phenylmethylsulfonyl fluoride protein-tyrosine phosphatase 1B soluble adenylyl cyclase streptolysin O N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine inositol 1,4,5-trisphosphate phosphate-buffered saline guanosine 5′-3-O-(thio)triphosphate glutathione S-transferase soluble NSF attachment protein receptors phosphatidylinositol-specific phospholipase C. In certain neurons, neuroendocrine and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis. Either cAMP-dependent protein kinase (PKA) or the exchange protein directly activated by cAMP (Epac) can be the targets of cAMP in the cAMP-regulated exocytosis. On the other hand, cAMP is the principal trigger of regulated secretion in various non-neuronal cells (5Seino S. Shibasaki T. Physiol. Rev. 2005; 85: 1303-1342Crossref PubMed Scopus (459) Google Scholar, 6Burgoyne R.D. Morgan A. Physiol. Rev. 2003; 83: 581-632Crossref PubMed Scopus (544) Google Scholar, 7Szaszák M. Christian F. Rosenthal W. Klussmann E. Cell. Signal. 2008; 20: 590-601Crossref PubMed Scopus (49) Google Scholar). Likewise, an elevation of cAMP alone is sufficient to trigger exocytosis in human sperm. Moreover, calcium relies on endogenous cAMP to accomplish acrosomal release, and it does so through a PKA-insensitive pathway involving Epac. The stimulation of endogenous Epac by the selective cAMP analogue 8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) is sufficient to trigger the AR even in the absence of extracellular calcium. Furthermore, when Epac is sequestered with specific antibodies, cAMP, calcium (8Branham M.T. Mayorga L.S. Tomes C.N. J. Biol. Chem. 2006; 281: 8656-8666Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and recombinant Rab3A (this study) are unable to elicit exocytosis. Epac1 and Epac2 are multidomain proteins that consist of an N-terminal regulatory region and a C-terminal catalytic region (9Bos J.L. Trends Biochem. Sci. 2006; 31: 680-686Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 10Holz G.G. Kang G. Harbeck M. Roe M.W. Chepurny O.G. J. Physiol. 2006; 577: 5-15Crossref PubMed Scopus (229) Google Scholar, 11Roscioni S.S. Elzinga C.R. Schmidt M. Naunyn Schmiedebergs Arch. Pharmacol. 2008; 377: 345-357Crossref PubMed Scopus (122) Google Scholar). The regulatory domain harbors the cAMP-binding site, which auto-inhibits the catalytic activity in the absence of cAMP (12de Rooij J. Rehmann H. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 2000; 275: 20829-20836Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 13Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14Harper S.M. Wienk H. Wechselberger R.W. Bos J.L. Boelens R. Rehmann H. J. Biol. Chem. 2008; 283: 6501-6508Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 15Rehmann H. Arias-Palomo E. Hadders M.A. Schwede F. Llorca O. Bos J.L. Nature. 2008; 455: 124-127Crossref PubMed Scopus (146) Google Scholar). The catalytic portion bears a guanine-nucleotide exchange factor (GEF) activity specific for Rap1 and Rap2 (16de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1624) Google Scholar, 17Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1175) Google Scholar). Like all small G proteins, Raps cycle between an inactive GDP-bound and an active GTP-bound conformation. The GDP-GTP cycle is regulated by GEFs that induce the release of the bound GDP to be replaced by the more abundant GTP and by GTPase-activating proteins that coax the intrinsic GTPase activity to rapidly hydrolyze bound GTP, returning the G proteins to the inactive GDP-bound state (18Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acid Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar, 19Zheng Y. Quilliam L.A. EMBO Rep. 2003; 4: 463-468Crossref PubMed Scopus (16) Google Scholar). Most small G proteins are linked to biological membranes via lipid modifications at their C terminus; for instance, Rap2A is farnesylated, and Rap1A/B, Rap2B, and Rabs are geranylgeranylated (20Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 21Stork P.J. Trends Biochem. Sci. 2003; 28: 267-275Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Guanine nucleotide dissociation inhibitors (GDIs) remove Rabs from membranes by sequestration of their lipid tails (22Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1314) Google Scholar). Extracellular stimuli often result in the activation of cellular adenylate cyclases and an increase in cAMP levels. By serving as a cAMP-binding protein with intrinsic GEF activity, Epac couples cAMP production to a variety of Rap-mediated processes such as the control of cell adhesion and cell-cell junction formation, water resorption, cell differentiation, inflammatory processes, etc. (9Bos J.L. Trends Biochem. Sci. 2006; 31: 680-686Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 10Holz G.G. Kang G. Harbeck M. Roe M.W. Chepurny O.G. J. Physiol. 2006; 577: 5-15Crossref PubMed Scopus (229) Google Scholar, 11Roscioni S.S. Elzinga C.R. Schmidt M. Naunyn Schmiedebergs Arch. Pharmacol. 2008; 377: 345-357Crossref PubMed Scopus (122) Google Scholar). Many are the effectors of Epac and Epac-Rap signaling. Of particular interest to us is the observation that Epac stimulates phospholipase Cϵ (PLCϵ) through the activation of Rap1 and -2, resulting in IP3-mediated release of calcium from internal stores (23Schmidt M. Evellin S. Weernink P.A. von Dorp F. Rehmann H. Lomasney J.W. Jakobs K.H. Nat. Cell Biol. 2001; 3: 1020-1024Crossref PubMed Scopus (282) Google Scholar, 24Oestreich E.A. Wang H. Malik S. Kaproth-Joslin K.A. Blaxall B.C. Kelley G.G. Dirksen R.T. Smrcka A.V. J. Biol. Chem. 2007; 282: 5488-5495Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). PLCϵ is an unusual enzyme with two catalytic activities as follows: the typical phosphatidylinositol 4,5-bisphosphate hydrolyzing PLC activity plus a Rap-GEF activity. Thus, PLCϵ acts both downstream and upstream of Ras-like GTPases, perhaps to guarantee sustained Rap signaling (25Bunney T.D. Katan M. Trends Cell Biol. 2006; 16: 640-648Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). During membrane fusion, Rab proteins direct the recognition and physical attachments of the compartments that are going to fuse (26Gonzalez Jr., L. Scheller R.H. Cell. 1999; 96: 755-758Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 27Pfeffer S.R. Nat. Cell Biol. 1999; 1: E17-E22Crossref PubMed Scopus (361) Google Scholar). This association, or tethering, represents one of the earliest known events in membrane fusion and is accomplished through the recruitment of tethering factors. Rab3A localizes to vesicles and secretory granules and is one of the isoforms directly implicated in regulated exocytosis of neurotransmitters and hormones (28Lang T. Jahn R. Handb. Exp. Pharmacol. 2008; 184: 107-127Crossref PubMed Scopus (98) Google Scholar). Rab3A interacts in a GTP-dependent manner with at least two effector proteins, rabphilin and Rim (29Regazzi R. Regazzi R. Molecular Mechanisms of Exocytosis. Landes Bioscience, Austin, TX2007: 28-41Crossref Google Scholar, 30Handley M.T. Haynes L.P. Burgoyne R.D. J. Cell Sci. 2007; 120: 973-984Crossref PubMed Scopus (58) Google Scholar, 31Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2708) Google Scholar). Rab3A is present in the acrosomal region of human (32Yunes R. Michaut M. Tomes C. Mayorga L.S. Biol. Reprod. 2000; 62: 1084-1089Crossref PubMed Scopus (82) Google Scholar), rat (33Iida H. Yoshinaga Y. Tanaka S. Toshimori K. Mori T. Dev. Biol. 1999; 211: 144-155Crossref PubMed Scopus (76) Google Scholar), and mouse sperm (34Ward C.R. Faundes D. Foster J.A. Mol. Reprod. Dev. 1999; 53: 413-421Crossref PubMed Scopus (42) Google Scholar). Rab3A (full-length recombinant protein or a synthetic peptide corresponding to the effector domain) stimulates human (32Yunes R. Michaut M. Tomes C. Mayorga L.S. Biol. Reprod. 2000; 62: 1084-1089Crossref PubMed Scopus (82) Google Scholar, 35Lopez C.I. Belmonte S.A. De Blas G.A. Mayorga L.S. FASEB J. 2007; 21: 4121-4130Crossref PubMed Scopus (42) Google Scholar) and ram (36Garde J. Roldan E.R. FEBS Lett. 1996; 391: 263-268Crossref PubMed Scopus (38) Google Scholar) and inhibits rat sperm AR (33Iida H. Yoshinaga Y. Tanaka S. Toshimori K. Mori T. Dev. Biol. 1999; 211: 144-155Crossref PubMed Scopus (76) Google Scholar). Rab3A is required for the AR triggered by calcium (37Belmonte S.A. López C.I. Roggero C.M. De Blas G.A. Tomes C.N. Mayorga L.S. Dev. Biol. 2005; 285: 393-408Crossref PubMed Scopus (47) Google Scholar, 38De Blas G.A. Roggero C.M. Tomes C.N. Mayorga L.S. PLoS. Biol. 2005; 3: e323Crossref PubMed Scopus (105) Google Scholar) and cAMP (8Branham M.T. Mayorga L.S. Tomes C.N. J. Biol. Chem. 2006; 281: 8656-8666Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Epac is a multifunctional protein in which cAMP exerts its effects not only by promoting the exchange of GDP for GTP on Rap but also by allosterically regulating other molecules (10Holz G.G. Kang G. Harbeck M. Roe M.W. Chepurny O.G. J. Physiol. 2006; 577: 5-15Crossref PubMed Scopus (229) Google Scholar). In exocytosis for instance, a number of Rap-independent, Epac-linked signaling pathways have been described. They include the interaction of Epac2 with Rim2 (39Ozaki N. Shibasaki T. Kashima Y. Miki T. Takahashi K. Ueno H. Sunaga Y. Yano H. Matsuura Y. Iwanaga T. Takai Y. Seino S. Nat. Cell Biol. 2000; 2: 805-811Crossref PubMed Scopus (399) Google Scholar) and the Rim2-related protein Piccolo (40Fujimoto K. Shibasaki T. Yokoi N. Kashima Y. Matsumoto M. Sasaki T. Tajima N. Iwanaga T. Seino S. J. Biol. Chem. 2002; 277: 50497-50502Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Epac2 also stimulates exocytosis by interacting with SUR1 (41Eliasson L. Ma X. Renström E. Barg S. Berggren P.O. Galvanovskis J. Gromada J. Jing X. Lundquist I. Salehi A. Sewing S. Rorsman P. J. Gen. Physiol. 2003; 121: 181-197Crossref PubMed Scopus (217) Google Scholar). Finally, Epac2 controls ryanodine-sensitive calcium channels that are involved in calcium-induced calcium release (CICR) from internal stores in insulin-secreting cells (42Kang G. Joseph J.W. Chepurny O.G. Monaco M. Wheeler M.B. Bos J.L. Schwede F. Genieser H.G. Holz G.G. J. Biol. Chem. 2003; 278: 8279-8285Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). In this study, we piece together the analysis of two phenomena as follows: calcium mobilization and protein-protein interactions preceding exocytosis. To the best of our knowledge, this constitutes the first integrated molecular model that includes both the assembly of the fusion and intravesicular calcium release protein machineries during regulated exocytosis. By enquiring further into the signaling pathways operating during sperm exocytosis, we have found more players than previously suspected, and we discovered that the key components of these cascades are not arranged in a linear sequence. Epac sits at a central point of the signaling cascade after which the exocytotic pathway splits into two limbs as follows: one that assembles the fusion machinery into place, and another that elicits the release of calcium from the acrosome; both need to act in concert to achieve exocytosis. Our results identify Rab3A for the first time as a downstream target for Epac and place this small GTPase as an early component of the “fusion machinery” branch of the pathway. They also show that Epac stimulates the exchange of GDP for GTP on Rap1 and that this protein, as well as a PLC, drives intracellular calcium mobilization. Finally, our data reveal that a soluble adenylyl cyclase (sAC) (43Steegborn C. Litvin T.N. Levin L.R. Buck J. Wu H. Nat. Struct. Mol. Biol. 2005; 12: 32-37Crossref PubMed Scopus (137) Google Scholar, 44Kamenetsky M. Middelhaufe S. Bank E.M. Levin L.R. Buck J. Steegborn C. J. Mol. Biol. 2006; 362: 623-639Crossref PubMed Scopus (252) Google Scholar) synthesizes the cAMP that activates Epac. Again, we believe that this is the first report linking sAC to an exocytotic event. Recombinant streptolysin O (SLO) was obtained from Dr. Bhakdi (University of Mainz, Mainz, Germany). Spermatozoa were cultured in human tubal fluid media (as formulated by Irvine Scientific, Santa Ana, CA) supplemented with 0.5% bovine serum albumin (HTF media). The rabbit polyclonal antibodies to Rap1A/B and Rab3A (purified IgG) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The rabbit polyclonal anti-NSF (whole serum) was from Synaptic Systems (Göttingen, Germany), and rabbit polyclonal antibody to Rab11 was from Invitrogen. The rabbit polyclonal antibodies against Epac were generated by Genemed Synthesis, Inc. (San Francisco), using the synthetic peptide LREDNCHFLRVDK, and affinity-purified on immobilized Epac peptide (8Branham M.T. Mayorga L.S. Tomes C.N. J. Biol. Chem. 2006; 281: 8656-8666Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Anti-Rap 1A/B rabbit polyclonal antibodies were raised against the peptide EDERVVGKEQGQNLC and affinity-purified on immobilized peptide (GenScript Corp., Piscataway, NJ). Horseradish peroxidase-conjugated goat anti-rabbit IgG (anti-γ chain) was from Jackson ImmunoResearch (West Grove, PA). Glutathione S-transferase (GST)-tagged, human recombinant cAMP-specific phosphodiesterase 4D (PDE), and Rap1A were from Abnova Corp. (NeiHu, Taipei, Taiwan). KH7 was purchased from ChemDiv, Inc. (San Diego). 8-pCPT-2′-O- Me-cAMP, N6-benzoyladenosine-3′,5′-cyclic monophosphate (6-Bnz-cAMP), and 2′,3′-O-(N′-methylanthraniloyl)-GDP(mGDP) were from Biolog-Life Science Institute (Bremen, Germany). U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) and the inactive analogue U73343 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione) were from Biomol International L.P. (Plymouth Meeting, PA). Adenophostin A, hexasodium salt, and 2-aminoethoxydiphenyl borate (2-APB) from Calbiochem were purchased from Merck Química Argentina S.A.I.C. (Buenos Aires, Argentina). O-Nitrophenyl EGTA-acetoxymethyl ester (NP-EGTA-AM) and Fluo-3-AM were purchased from Molecular Probes (Eugene, OR). Prestained molecular weight markers were from Boston BioProducts, Inc. (Worcester, MA). Glutathione-Sepharose and nickel-nitrilotriacetic acid-agarose were from GE Healthcare. All other chemicals were purchased from Sigma, Genbiotech, or Tecnolab (all from Buenos Aires, Argentina). Plasmid pGEX-2T containing the cDNA-encoding human Rab3A was provided by Dr. P. Stahl (Washington University, St. Louis, MO). The Rap1-GTP binding cassette Ral-GDS-RBD fused to GST (45van Triest M. de Rooij J. Bos J.L. Methods Enzymol. 2001; 333: 343-348Crossref PubMed Scopus (75) Google Scholar) was a kind gift from Dr. O. Coso (Universidad de Buenos Aires, Buenos Aires, Argentina). The Rab3-GTP binding cassette RIM-RBD fused to GST (46Coppola T. Perret-Menoud V. Gattesco S. Magnin S. Pombo I. Blank U. Regazzi R. Biochem. J. 2002; 362: 273-279Crossref PubMed Scopus (47) Google Scholar) was generously provided by Dr. R. Regazzi (University of Lausanne, Lausanne, Switzerland). cDNA encoding GDI-α was a kind gift from Dr. Y. Takai (Osaka University, Osaka, Japan). The expression plasmid pGEX-3X (GE Healthcare) encoding GST fused to amino acids 1–321 of PTP1B bearing the substrate-trapping mutation D181A was kindly provided by Dr. N. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Plasmid pAC21 encoding a His6-tagged phosphomimetic mutant of NSF (NSF Y83E) was a gift from Dr. N. Bottini (The Burnham Institute, La Jolla, CA). The Epac1 construct is derived from human cDNA and the Epac2 construct from murine cDNA. Epac1 (amino acids 149–881) and Epac2 (amino acids 280–993) were expressed as GST fusion proteins from the pGEX-4T2 vector in the Escherichia coli strain CK600K as described previously (47Kraemer A. Rehmann H.R. Cool R.H. Theiss C. de Rooij J. Bos J.L. Wittinghofer A. J. Mol. Biol. 2001; 306: 1167-1177Crossref PubMed Scopus (58) Google Scholar). GST-Rab3A was expressed in E. coli BL21 (Stratagene) as described (48Roggero C.M. De Blas G.A. Dai H. Tomes C.N. Rizo J. Mayorga L.S. J. Biol. Chem. 2007; 282: 26335-26343Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). GST-Ral-GDS-RBD, GST-RIM, GST-PTP1B D181A, GST-Rap1A, and GST-GDI were transformed into E. coli BL21, and protein expression was induced with isopropyl β-d-thio-galactoside (0.2 mm for the first two proteins and 0.1 mm for the rest) for 16 h at 22 °C. All GST-fused recombinant proteins were purified on glutathione-Sepharose following standard procedures, except GST-Ral-GDS-RBD and GST-RIM, where bacterial lysates were frozen until use. His6-NSF Y83E was expressed and purified on nickel-nitrilotriacetic acid-agarose as described for wild type NSF (49Zarelli V.E. Ruete M.C. Roggero C.M. Mayorga L.S. Tomes C.N. J. Biol. Chem. 2009; 284: 10491-10503Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Purified, recombinant His6-Rab3A (35Lopez C.I. Belmonte S.A. De Blas G.A. Mayorga L.S. FASEB J. 2007; 21: 4121-4130Crossref PubMed Scopus (42) Google Scholar) was a kind gift from Dr. C. López (IHEM, Mendoza, Argentina). Rab3A proteins were prenylated for all but the in vitro activation experiments and loaded with the appropriate guanosine nucleotides. Recombinant protein concentrations were determined by the Bio-Rad protein assay in 96-well microplates. Bovine serum albumin was used as a standard, and the results were quantified on a 3550 Microplate Reader (Bio-Rad). Experiments were performed as described (50van den Berghe N. Cool R.H. Horn G. Wittinghofer A. Oncogene. 1997; 15: 845-850Crossref PubMed Scopus (87) Google Scholar, 51Rehmann H. Methods Enzymol. 2006; 407: 159-173Crossref PubMed Scopus (35) Google Scholar), using Rab3A in addition to Rap1. Purified Rab3A or Rap1 (200 nm, GST removed with thrombin) loaded with the fluorescent GDP analogue mGDP was incubated in the presence of 20 μm unlabeled GDP plus 150 nm purified Epac1 or Epac2. Cyclic AMP (100–500 μm) was added as indicated. The nucleotide exchange was measured in real time as decay in fluorescence using a Cary Eclipse spectrofluorometer (Varian, Varian B.V., Middelburg, The Netherlands). The decay is caused by the release of protein-bound mGDP, which shows higher fluorescence intensity in the hydrophobic environment of the protein than in the buffer solution. The decay in the fluorescence signal is equal to the rate of nucleotide dissociation, and nucleotide exchange should be accelerated in the presence of an active GEF. All data analysis, fitting, and plotting were done with the Grafit 3.0 program (Erithacus software). Human semen samples were obtained from normal healthy donors. Semen was allowed to liquify for 30–60 min at 37 °C. We used a swim-up protocol to isolate highly motile sperm. Sperm concentrations were adjusted to 5–10 × 106/ml before incubating for at least 2 h under capacitating conditions (HTF, 37 °C, 5% CO2, 95% air). For experiments involving intact sperm, we added increasing concentrations of KH7 and 10 μm A23187 or 5 μm progesterone sequentially and incubated for 10–15 min at 37 °C after each addition. For experiments involving permeabilized cells, washed spermatozoa were resuspended in cold PBS containing 2.1 units/ml SLO for 15 min at 4 °C. Cells were washed once with PBS and resuspended in ice-cold sucrose buffer (250 mm sucrose, 0.5 mm EGTA, 20 mm Hepes-K, pH 7) containing 2 mm dithiothreitol. We added inhibitors and stimulants sequentially as indicated in the figure legends and incubated for 10–15 min at 37 °C after each addition. Where indicated, we preloaded SLO-permeabilized sperm with NP-EGTA-AM before incubating with inhibitors and/or calcium, carrying out all procedures in the dark. Photolysis was induced after the last incubation by exposing twice (1 min each time) to an UV transilluminator and mixing after each exposure. Intact and permeabilized sperm were spotted on Teflon-printed slides, air-dried, and fixed/permeabilized in ice-cold methanol for 1 min. Acrosomal status was evaluated by staining with FITC-coupled Pisum sativum (FITC-PSA) (52Mendoza C. Carreras A. Moos J. Tesarik J. J. Reprod. Fertil. 1992; 95: 755-763Crossref PubMed Scopus (227) Google Scholar). At least 200 cells were scored using an upright Nikon microscope equipped with epifluorescence optics. Basal (no stimulation) and positive (0.5 mm CaCl2 corresponding to 10 μm free calcium estimated by MAXCHELATOR, a series of program(s) for determining the free metal concentration in the presence of chelators) controls were included in all experiments. Acrosomal exocytosis indexes were calculated by subtracting the number of spontaneously reacted spermatozoa from all values and expressing the results as a percentage of the AR observed in the positive control. Data were evaluated using one way analysis of variance. The Tukey-Kramer post hoc test was used for pairwise comparisons. Differences were considered significant at the p < 0.05 level. Capacitated sperm (10–50 × 106 cells) were washed twice and suspended in PBS. Sperm were treated with 100 μm 2-APB (an IP3-sensitive calcium channel blocker) to prevent cytosol/membrane loss due to exocytosis before adding the AR inducers. Alternatively, sperm were permeabilized with SLO and treated with 2-APB and 20 μg/ml anti-Rap1 antibodies (GenScript) or 1 μg of His6-Rab3A before challenging with 50 μm 8-pCPT-2′-O-Me-cAMP or 0.5 mm CaCl2. After 10 min of incubation at 37 °C, cells were lysed in GST pulldown buffer (200 mm NaCl, 2.5 mm MgCl2, 1% (v/v) Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1× protease inhibitor mixture (P2714, Sigma), and 50 mm Tris-HCl, pH 7.4) by sonication on ice (two times for 15 s). We let proteins diffuse into the lysis buffer for 15 min at 4 °C. These whole cell detergent extracts were clarified by centrifugation at 12,000 × g for 5 min and used immediately. Glutathione-Sepharose beads were washed twice with GST pulldown buffer and incubated with bacterial lysates containing GST-Ral-GDS-RBD or GST-RIM for 1 h at 4 °C under constant rocking. Beads were washed twice with PBS and once with GST pulldown buffer and used immediately. Twenty μl of glutathione-Sepharose containing 10 μg of the appropriate fusion protein was added to sperm lysates in a total volume of 0.6 ml and incubated by rotation at 4 °C for 30 min. The resin was recovered by centrifugation at 4 °C (5 min at 10,000 rpm) and washed three times with ice-cold GST pulldown buffer. The resin-bound fractions were resolved by SDS-PAGE, and cellular GTP-Rap1 and GTP-Rab3A levels were analyzed by immunoblotting as described later. Partition experiments were conducted following standard procedures (53Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar, 54Pryde J.G. Phillips J.H. Biochem. J. 1986; 233: 525-533Crossref PubMed Scopus (122) Google Scholar). Briefly, capacitated sperm (100 × 106 cells) were washed twice with cold PBS," @default.
• W2048096689 created "2016-06-24" @default.
• W2048096689 creator A5000837316 @default.
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• W2048096689 creator A5019139724 @default.
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• W2048096689 creator A5089226233 @default.
• W2048096689 date "2009-09-01" @default.
• W2048096689 modified "2023-10-01" @default.
• W2048096689 title "Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis" @default.
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