Rab3D redistribution and function in rat parotid acini
An Erratum has been published for this article in Journal of Cellular Physiology 199: 316, 2004. Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cy...
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| Veröffentlicht in: | Journal of cellular physiology 2003-12, Vol.197 (3), p.400-408 |
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| creator | Ngyen, Danielle Jones, Antoinette Ojakian, George K. Raffaniello, Robert D. |
| description | An Erratum has been published for this article in Journal of Cellular Physiology 199: 316, 2004.
Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.
J. Cell. Physiol. 197: 400–408, 2003© 2003 Wiley‐Liss, Inc. |
| doi_str_mv | 10.1002/jcp.10373 |
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Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.
J. Cell. Physiol. 197: 400–408, 2003© 2003 Wiley‐Liss, Inc.</description><identifier>ISSN: 0021-9541</identifier><identifier>EISSN: 1097-4652</identifier><identifier>DOI: 10.1002/jcp.10373</identifier><identifier>PMID: 14566969</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>Amylases - metabolism ; Animals ; Calcium - metabolism ; Calcium - pharmacology ; Cell Compartmentation - physiology ; Cell Membrane - metabolism ; Cyclic AMP - metabolism ; Cyclic AMP - pharmacology ; Cytoplasm - metabolism ; Epithelial Cells - metabolism ; Epithelial Cells - physiology ; Male ; Mutation - physiology ; Parotid Gland - metabolism ; Parotid Gland - physiology ; Protein Binding - physiology ; rab3 GTP-Binding Proteins - genetics ; rab3 GTP-Binding Proteins - metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins - metabolism ; Secretory Vesicles - metabolism</subject><ispartof>Journal of cellular physiology, 2003-12, Vol.197 (3), p.400-408</ispartof><rights>Copyright © 2003 Wiley‐Liss, Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4253-7b69a628fd961798f71cd8e29f646418baa77e4334ea70d4ce0df699dc47dadb3</citedby><cites>FETCH-LOGICAL-c4253-7b69a628fd961798f71cd8e29f646418baa77e4334ea70d4ce0df699dc47dadb3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fjcp.10373$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fjcp.10373$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27923,27924,45573,45574</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14566969$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ngyen, Danielle</creatorcontrib><creatorcontrib>Jones, Antoinette</creatorcontrib><creatorcontrib>Ojakian, George K.</creatorcontrib><creatorcontrib>Raffaniello, Robert D.</creatorcontrib><title>Rab3D redistribution and function in rat parotid acini</title><title>Journal of cellular physiology</title><addtitle>J. Cell. Physiol</addtitle><description>An Erratum has been published for this article in Journal of Cellular Physiology 199: 316, 2004.
Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.
J. Cell. Physiol. 197: 400–408, 2003© 2003 Wiley‐Liss, Inc.</description><subject>Amylases - metabolism</subject><subject>Animals</subject><subject>Calcium - metabolism</subject><subject>Calcium - pharmacology</subject><subject>Cell Compartmentation - physiology</subject><subject>Cell Membrane - metabolism</subject><subject>Cyclic AMP - metabolism</subject><subject>Cyclic AMP - pharmacology</subject><subject>Cytoplasm - metabolism</subject><subject>Epithelial Cells - metabolism</subject><subject>Epithelial Cells - physiology</subject><subject>Male</subject><subject>Mutation - physiology</subject><subject>Parotid Gland - metabolism</subject><subject>Parotid Gland - physiology</subject><subject>Protein Binding - physiology</subject><subject>rab3 GTP-Binding Proteins - genetics</subject><subject>rab3 GTP-Binding Proteins - metabolism</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Secretory Vesicles - metabolism</subject><issn>0021-9541</issn><issn>1097-4652</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kLFOwzAQhi0EoqUw8AIoExJDqB07djyiAgVUoCog2CzHdiRDmgQ7EfTtMU2BienudN_90n0AHCJ4iiBMxq-qCQ1meAsMEeQsJjRNtsEw7FDMU4IGYM_7Vwgh5xjvggEiKaWc8iGgC5nj88gZbX3rbN61tq4iWemo6Cq1HmwVOdlGjXR1a3Ukla3sPtgpZOnNwaaOwNPlxePkKp7dT68nZ7NYkSTFMcsplzTJCs0pYjwrGFI6MwkvKKEEZbmUjBmCMTGSQU2UgbqgnGtFmJY6xyNw3Oc2rn7vjG_F0nplylJWpu68YCjJSHgkgCc9qFztvTOFaJxdSrcSCIpvSSJIEmtJgT3ahHb50ug_cmMlAOMe-LClWf2fJG4m85_IuL8IFs3n74V0b4IyzFLxfDcV6fx28bCAWLzgL8KVfyA</recordid><startdate>200312</startdate><enddate>200312</enddate><creator>Ngyen, Danielle</creator><creator>Jones, Antoinette</creator><creator>Ojakian, George K.</creator><creator>Raffaniello, Robert D.</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><scope>BSCLL</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></search><sort><creationdate>200312</creationdate><title>Rab3D redistribution and function in rat parotid acini</title><author>Ngyen, Danielle ; Jones, Antoinette ; Ojakian, George K. ; Raffaniello, Robert D.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4253-7b69a628fd961798f71cd8e29f646418baa77e4334ea70d4ce0df699dc47dadb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Amylases - metabolism</topic><topic>Animals</topic><topic>Calcium - metabolism</topic><topic>Calcium - pharmacology</topic><topic>Cell Compartmentation - physiology</topic><topic>Cell Membrane - metabolism</topic><topic>Cyclic AMP - metabolism</topic><topic>Cyclic AMP - pharmacology</topic><topic>Cytoplasm - metabolism</topic><topic>Epithelial Cells - metabolism</topic><topic>Epithelial Cells - physiology</topic><topic>Male</topic><topic>Mutation - physiology</topic><topic>Parotid Gland - metabolism</topic><topic>Parotid Gland - physiology</topic><topic>Protein Binding - physiology</topic><topic>rab3 GTP-Binding Proteins - genetics</topic><topic>rab3 GTP-Binding Proteins - metabolism</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Secretory Vesicles - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ngyen, Danielle</creatorcontrib><creatorcontrib>Jones, Antoinette</creatorcontrib><creatorcontrib>Ojakian, George K.</creatorcontrib><creatorcontrib>Raffaniello, Robert D.</creatorcontrib><collection>Istex</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><jtitle>Journal of cellular physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ngyen, Danielle</au><au>Jones, Antoinette</au><au>Ojakian, George K.</au><au>Raffaniello, Robert D.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Rab3D redistribution and function in rat parotid acini</atitle><jtitle>Journal of cellular physiology</jtitle><addtitle>J. Cell. Physiol</addtitle><date>2003-12</date><risdate>2003</risdate><volume>197</volume><issue>3</issue><spage>400</spage><epage>408</epage><pages>400-408</pages><issn>0021-9541</issn><eissn>1097-4652</eissn><abstract>An Erratum has been published for this article in Journal of Cellular Physiology 199: 316, 2004.
Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.
J. Cell. Physiol. 197: 400–408, 2003© 2003 Wiley‐Liss, Inc.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>14566969</pmid><doi>10.1002/jcp.10373</doi><tpages>9</tpages></addata></record> |
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| subjects | Amylases - metabolism Animals Calcium - metabolism Calcium - pharmacology Cell Compartmentation - physiology Cell Membrane - metabolism Cyclic AMP - metabolism Cyclic AMP - pharmacology Cytoplasm - metabolism Epithelial Cells - metabolism Epithelial Cells - physiology Male Mutation - physiology Parotid Gland - metabolism Parotid Gland - physiology Protein Binding - physiology rab3 GTP-Binding Proteins - genetics rab3 GTP-Binding Proteins - metabolism Rats Rats, Sprague-Dawley Recombinant Fusion Proteins - metabolism Secretory Vesicles - metabolism |
| title | Rab3D redistribution and function in rat parotid acini |
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