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 byp...
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Veröffentlicht in: | The Journal of biological chemistry 2009-09, Vol.284 (37), p.24825-24839 |
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creator | Branham, María T. Bustos, Matías A. De Blas, Gerardo A. Rehmann, Holger Zarelli, Valeria E.P. Treviño, Claudia L. Darszon, Alberto Mayorga, Luis S. Tomes, Claudia N. |
description | 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. |
doi_str_mv | 10.1074/jbc.M109.015362 |
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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.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M109.015362</identifier><identifier>PMID: 19546222</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Acrosome Reaction ; Apolipoprotein A-V ; Apolipoproteins A - metabolism ; Calcium - metabolism ; Exocytosis - physiology ; Fertilization ; GTP-Binding Proteins - metabolism ; Guanine Nucleotide Exchange Factors - metabolism ; Guanosine Triphosphate - metabolism ; Humans ; Male ; Mechanisms of Signal Transduction ; Models, Biological ; Protein Structure, Tertiary ; Recombinant Proteins - chemistry ; Recombinant Proteins - metabolism ; Spermatozoa - metabolism ; Telomere-Binding Proteins - metabolism</subject><ispartof>The Journal of biological chemistry, 2009-09, Vol.284 (37), p.24825-24839</ispartof><rights>2009 © 2009 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2009 by The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c560t-2c70e14ca86b254fde03e92dd9cdff24715eea558b15d98848a5f914145b8fc53</citedby><cites>FETCH-LOGICAL-c560t-2c70e14ca86b254fde03e92dd9cdff24715eea558b15d98848a5f914145b8fc53</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757186/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757186/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19546222$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Branham, María T.</creatorcontrib><creatorcontrib>Bustos, Matías A.</creatorcontrib><creatorcontrib>De Blas, Gerardo A.</creatorcontrib><creatorcontrib>Rehmann, Holger</creatorcontrib><creatorcontrib>Zarelli, Valeria E.P.</creatorcontrib><creatorcontrib>Treviño, Claudia L.</creatorcontrib><creatorcontrib>Darszon, Alberto</creatorcontrib><creatorcontrib>Mayorga, Luis S.</creatorcontrib><creatorcontrib>Tomes, Claudia N.</creatorcontrib><title>Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>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.</description><subject>Acrosome Reaction</subject><subject>Apolipoprotein A-V</subject><subject>Apolipoproteins A - metabolism</subject><subject>Calcium - metabolism</subject><subject>Exocytosis - physiology</subject><subject>Fertilization</subject><subject>GTP-Binding Proteins - metabolism</subject><subject>Guanine Nucleotide Exchange Factors - metabolism</subject><subject>Guanosine Triphosphate - metabolism</subject><subject>Humans</subject><subject>Male</subject><subject>Mechanisms of Signal Transduction</subject><subject>Models, Biological</subject><subject>Protein Structure, Tertiary</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - metabolism</subject><subject>Spermatozoa - metabolism</subject><subject>Telomere-Binding Proteins - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kE1v1DAQhiMEokvhzA1yQNyy9fgjsS9Iq2ppkYpAlErcLMeZbFxl48X2LvTf41VWfBzwxZb8zDszT1G8BLIE0vCL-9YuPwJRSwKC1fRRsQAiWcUEfHtcLAihUCkq5FnxLMZ7kg9X8LQ4AyV4TSldFNfrnbHlyiZ3MAljmQYsb7dmHMur8nPwCd0Uyy9mB6WZuvxo2apMPhcMDg9Yrn96-5B8dPF58aQ3Y8QXp_u8uHu__np5Xd18uvpwubqprKhJqqhtCAK3RtYtFbzvkDBUtOuU7fqe8gYEohFCtiA6JSWXRvQKOHDRyt4Kdl68m3N3-3aLncUpBTPqXXBbEx60N07_-zO5QW_8QdNGNCDrHPD2FBD89z3GpLcuWhxHM6HfR103NWNckQxezKANPsaA_e8mQPTRvs729dG-nu3nild_z_aHP-nOwJsZGNxm-OEC6tZ5O-BWU8k1azTlkh53fD1jvfHabIKL-u6WEmAE6kYIOC6hZgKz6oPDoKN1OFnscqhNuvPuv1P-AnqmqFM</recordid><startdate>20090911</startdate><enddate>20090911</enddate><creator>Branham, María T.</creator><creator>Bustos, Matías A.</creator><creator>De Blas, Gerardo A.</creator><creator>Rehmann, Holger</creator><creator>Zarelli, Valeria E.P.</creator><creator>Treviño, Claudia L.</creator><creator>Darszon, Alberto</creator><creator>Mayorga, Luis S.</creator><creator>Tomes, Claudia N.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20090911</creationdate><title>Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis</title><author>Branham, María T. ; Bustos, Matías A. ; De Blas, Gerardo A. ; Rehmann, Holger ; Zarelli, Valeria E.P. ; Treviño, Claudia L. ; Darszon, Alberto ; Mayorga, Luis S. ; Tomes, Claudia N.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c560t-2c70e14ca86b254fde03e92dd9cdff24715eea558b15d98848a5f914145b8fc53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Acrosome Reaction</topic><topic>Apolipoprotein A-V</topic><topic>Apolipoproteins A - metabolism</topic><topic>Calcium - metabolism</topic><topic>Exocytosis - physiology</topic><topic>Fertilization</topic><topic>GTP-Binding Proteins - metabolism</topic><topic>Guanine Nucleotide Exchange Factors - metabolism</topic><topic>Guanosine Triphosphate - metabolism</topic><topic>Humans</topic><topic>Male</topic><topic>Mechanisms of Signal Transduction</topic><topic>Models, Biological</topic><topic>Protein Structure, Tertiary</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - metabolism</topic><topic>Spermatozoa - metabolism</topic><topic>Telomere-Binding Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Branham, María T.</creatorcontrib><creatorcontrib>Bustos, Matías A.</creatorcontrib><creatorcontrib>De Blas, Gerardo A.</creatorcontrib><creatorcontrib>Rehmann, Holger</creatorcontrib><creatorcontrib>Zarelli, Valeria E.P.</creatorcontrib><creatorcontrib>Treviño, Claudia L.</creatorcontrib><creatorcontrib>Darszon, Alberto</creatorcontrib><creatorcontrib>Mayorga, Luis S.</creatorcontrib><creatorcontrib>Tomes, Claudia N.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Branham, María T.</au><au>Bustos, Matías A.</au><au>De Blas, Gerardo A.</au><au>Rehmann, Holger</au><au>Zarelli, Valeria E.P.</au><au>Treviño, Claudia L.</au><au>Darszon, Alberto</au><au>Mayorga, Luis S.</au><au>Tomes, Claudia N.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2009-09-11</date><risdate>2009</risdate><volume>284</volume><issue>37</issue><spage>24825</spage><epage>24839</epage><pages>24825-24839</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>19546222</pmid><doi>10.1074/jbc.M109.015362</doi><tpages>15</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Acrosome Reaction Apolipoprotein A-V Apolipoproteins A - metabolism Calcium - metabolism Exocytosis - physiology Fertilization GTP-Binding Proteins - metabolism Guanine Nucleotide Exchange Factors - metabolism Guanosine Triphosphate - metabolism Humans Male Mechanisms of Signal Transduction Models, Biological Protein Structure, Tertiary Recombinant Proteins - chemistry Recombinant Proteins - metabolism Spermatozoa - metabolism Telomere-Binding Proteins - metabolism |
title | Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis |
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