Dynamics of receptor/G protein coupling in living cells
The interaction of activated G protein‐coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)‐based assay, we measure directly and in living cells the interaction of YFP‐labeled α 2A ‐adrenergic receptors with CFP‐labeled...
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| Veröffentlicht in: | The EMBO journal 2005-12, Vol.24 (23), p.4106-4114 |
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| creator | Hein, Peter Frank, Monika Hoffmann, Carsten Lohse, Martin J Bünemann, Moritz |
| description | The interaction of activated G protein‐coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)‐based assay, we measure directly and in living cells the interaction of YFP‐labeled α
2A
‐adrenergic receptors with CFP‐labeled G proteins. Upon agonist stimulation, a small, concentration‐dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were |
| doi_str_mv | 10.1038/sj.emboj.7600870 |
| format | Article |
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2A
‐adrenergic receptors with CFP‐labeled G proteins. Upon agonist stimulation, a small, concentration‐dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Gα. Simultaneously recorded G protein‐regulated inwardly rectifying K
+
channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Gα mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild‐type G proteins interacts with the activated receptor at any time. Our data suggest that α
2A
‐adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.</description><identifier>ISSN: 0261-4189</identifier><identifier>EISSN: 1460-2075</identifier><identifier>DOI: 10.1038/sj.emboj.7600870</identifier><identifier>PMID: 16292347</identifier><identifier>CODEN: EMJODG</identifier><language>eng</language><publisher>Chichester, UK: John Wiley & Sons, Ltd</publisher><subject>Adrenergic alpha-2 Receptor Agonists ; Bacterial Proteins - metabolism ; Cell Line ; Cell Membrane - metabolism ; EMBO37 ; Energy transfer ; Fluorescence ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; FRET ; G protein ; GIRK ; GTP-Binding Proteins - agonists ; GTP-Binding Proteins - metabolism ; Humans ; Kinetics ; Luminescent Proteins - metabolism ; Proteins ; Receptors, Adrenergic, alpha-2 - metabolism ; Receptors, G-Protein-Coupled - agonists ; Receptors, G-Protein-Coupled - metabolism ; Resonance ; Staining and Labeling ; α2A receptors</subject><ispartof>The EMBO journal, 2005-12, Vol.24 (23), p.4106-4114</ispartof><rights>European Molecular Biology Organization 2005</rights><rights>Copyright © 2005 European Molecular Biology Organization</rights><rights>Copyright Nature Publishing Group Dec 7, 2005</rights><rights>Copyright © 2005, European Molecular Biology Organization 2005</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5760-5013e50485e158d87442f4b7454401a0d562eb904e803a0a8f18e7bcd5e4a2a13</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1356310/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1356310/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,1411,1427,27901,27902,41096,42165,45550,45551,46384,46808,51551,53766,53768</link.rule.ids><linktorsrc>$$Uhttps://doi.org/10.1038/sj.emboj.7600870$$EView_record_in_Springer_Nature$$FView_record_in_$$GSpringer_Nature</linktorsrc><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16292347$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hein, Peter</creatorcontrib><creatorcontrib>Frank, Monika</creatorcontrib><creatorcontrib>Hoffmann, Carsten</creatorcontrib><creatorcontrib>Lohse, Martin J</creatorcontrib><creatorcontrib>Bünemann, Moritz</creatorcontrib><title>Dynamics of receptor/G protein coupling in living cells</title><title>The EMBO journal</title><addtitle>EMBO J</addtitle><addtitle>EMBO J</addtitle><description>The interaction of activated G protein‐coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)‐based assay, we measure directly and in living cells the interaction of YFP‐labeled α
2A
‐adrenergic receptors with CFP‐labeled G proteins. Upon agonist stimulation, a small, concentration‐dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Gα. Simultaneously recorded G protein‐regulated inwardly rectifying K
+
channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Gα mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild‐type G proteins interacts with the activated receptor at any time. Our data suggest that α
2A
‐adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.</description><subject>Adrenergic alpha-2 Receptor Agonists</subject><subject>Bacterial Proteins - metabolism</subject><subject>Cell Line</subject><subject>Cell Membrane - metabolism</subject><subject>EMBO37</subject><subject>Energy transfer</subject><subject>Fluorescence</subject><subject>Fluorescence Resonance Energy Transfer</subject><subject>Fluorescent Dyes</subject><subject>FRET</subject><subject>G protein</subject><subject>GIRK</subject><subject>GTP-Binding Proteins - agonists</subject><subject>GTP-Binding Proteins - metabolism</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Luminescent Proteins - metabolism</subject><subject>Proteins</subject><subject>Receptors, Adrenergic, alpha-2 - metabolism</subject><subject>Receptors, G-Protein-Coupled - agonists</subject><subject>Receptors, G-Protein-Coupled - metabolism</subject><subject>Resonance</subject><subject>Staining and Labeling</subject><subject>α2A receptors</subject><issn>0261-4189</issn><issn>1460-2075</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNp1UU2P0zAUtBCILYU7JxRx4Jbu81fsXJDo7lI-ClxAHC0nfS0OSRzsZKH_HpeWZUHi5JHezPPMG0IeU1hQ4Po8NgvsKt8sVAGgFdwhMyoKyBkoeZfMgBU0F1SXZ-RBjA0ASK3ofXJGC1YyLtSMqMt9bztXx8xvs4A1DqMP56tsCH5E12e1n4bW9bss4dZdH1CNbRsfkntb20Z8dHrn5NPLq48Xr_L1h9XrixfrvJbJUy6BcpQgtEQq9UYrIdhWVEpIIYBa2MiCYVWCQA3cgtVbqlFV9UaisMxSPifPj3uHqepwU2M_BtuaIbjOhr3x1pm_J737Ynb-2lAuC56ONCfPTguC_zZhHE3n4iGC7dFP0RRaa2DiQHz6D7HxU-hTOENLyQqZYiTSk9t2bnz8PmgilEfCd9fi_s8czKEwExvzqzBzKsxcvVu-OeGkpUdtTLJ-h-GWg__okyY_alwc8cfNfzZ8NYXiSprP71dmuVxrtnx7aTj_CUEmqTU</recordid><startdate>20051207</startdate><enddate>20051207</enddate><creator>Hein, Peter</creator><creator>Frank, Monika</creator><creator>Hoffmann, Carsten</creator><creator>Lohse, Martin J</creator><creator>Bünemann, Moritz</creator><general>John Wiley & Sons, Ltd</general><general>Nature Publishing Group UK</general><general>Blackwell Publishing Ltd</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>BKSAR</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M7N</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P64</scope><scope>PCBAR</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20051207</creationdate><title>Dynamics of receptor/G protein coupling in living cells</title><author>Hein, Peter ; Frank, Monika ; Hoffmann, Carsten ; Lohse, Martin J ; Bünemann, Moritz</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5760-5013e50485e158d87442f4b7454401a0d562eb904e803a0a8f18e7bcd5e4a2a13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Adrenergic alpha-2 Receptor Agonists</topic><topic>Bacterial Proteins - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The EMBO journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Hein, Peter</au><au>Frank, Monika</au><au>Hoffmann, Carsten</au><au>Lohse, Martin J</au><au>Bünemann, Moritz</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Dynamics of receptor/G protein coupling in living cells</atitle><jtitle>The EMBO journal</jtitle><stitle>EMBO J</stitle><addtitle>EMBO J</addtitle><date>2005-12-07</date><risdate>2005</risdate><volume>24</volume><issue>23</issue><spage>4106</spage><epage>4114</epage><pages>4106-4114</pages><issn>0261-4189</issn><eissn>1460-2075</eissn><coden>EMJODG</coden><abstract>The interaction of activated G protein‐coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)‐based assay, we measure directly and in living cells the interaction of YFP‐labeled α
2A
‐adrenergic receptors with CFP‐labeled G proteins. Upon agonist stimulation, a small, concentration‐dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Gα. Simultaneously recorded G protein‐regulated inwardly rectifying K
+
channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Gα mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild‐type G proteins interacts with the activated receptor at any time. Our data suggest that α
2A
‐adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.</abstract><cop>Chichester, UK</cop><pub>John Wiley & Sons, Ltd</pub><pmid>16292347</pmid><doi>10.1038/sj.emboj.7600870</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Adrenergic alpha-2 Receptor Agonists Bacterial Proteins - metabolism Cell Line Cell Membrane - metabolism EMBO37 Energy transfer Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Dyes FRET G protein GIRK GTP-Binding Proteins - agonists GTP-Binding Proteins - metabolism Humans Kinetics Luminescent Proteins - metabolism Proteins Receptors, Adrenergic, alpha-2 - metabolism Receptors, G-Protein-Coupled - agonists Receptors, G-Protein-Coupled - metabolism Resonance Staining and Labeling α2A receptors |
| title | Dynamics of receptor/G protein coupling in living cells |
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