Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers
Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusio...
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| Veröffentlicht in: | The Journal of biological chemistry 2012-07, Vol.287 (28), p.23604-23614 |
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| creator | Herrick-Davis, Katharine Grinde, Ellinor Lindsley, Tara Cowan, Ann Mazurkiewicz, Joseph E |
| description | Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric. |
| doi_str_mv | 10.1074/jbc.M112.350249 |
| format | Article |
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In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. 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In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.</description><subject>Animals</subject><subject>Cell Membrane - drug effects</subject><subject>Cell Membrane - metabolism</subject><subject>Cells, Cultured</subject><subject>Diffusion - drug effects</subject><subject>Endoplasmic Reticulum - metabolism</subject><subject>Fluorescence</subject><subject>Golgi Apparatus - metabolism</subject><subject>Green Fluorescent Proteins - chemistry</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>HEK293 Cells</subject><subject>Hippocampus - cytology</subject><subject>Humans</subject><subject>Luminescent Proteins - chemistry</subject><subject>Luminescent Proteins - genetics</subject><subject>Luminescent Proteins - metabolism</subject><subject>Microscopy, Confocal</subject><subject>Molecular Biophysics</subject><subject>Mutation</subject><subject>Neurons - cytology</subject><subject>Neurons - drug effects</subject><subject>Neurons - metabolism</subject><subject>Protein Multimerization</subject><subject>Protein Transport - drug effects</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Receptor, Serotonin, 5-HT2C - chemistry</subject><subject>Receptor, Serotonin, 5-HT2C - genetics</subject><subject>Receptor, Serotonin, 5-HT2C - metabolism</subject><subject>Serotonin - pharmacology</subject><subject>Spectrometry, Fluorescence - methods</subject><subject>Transfection</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkU1v1DAQhi1ERZfCmRvysRyy9UecDw5I1apQpKJeisQtcpzJxlXiCbazNPxSfg5mKQjm4hnPzPOOPYS84mzLWZlf3Ldm-4lzsZWKibx-QjacVTKTin95SjaMCZ7VQlWn5HkI9yxZXvNn5FQIVUtViQ35cTvaPU7gabDfgWJP4wA0gMeIzjqqsmHtPD6s0a9z1JN1QMWOnqvs-k7s3lAPBuaIPjkH0CN0tF1pPy7oIRhwBqhB72HU0aKjYQYTPQaD80q_2TjQefgllIoWF63b08GGiHuvJ6qdHtdgw1sKB9sdUX3SGXDCzqaBwxGAS6QTOjxepHSEmJpT8IKc9HoM8PLxPCOf31_d7a6zm9sPH3eXN9ksiiJmuWnLspai7WvViR5kUQrZd4LJSrCy6FneQm4qVRthdMGBtW1XmoqpQvJC16U8I-9-c-elnaBLb04DjM3s7aT92qC2zf8ZZ4dmj4dGypoVUiXA-SPA49cFQmwmm75uHLUDXELD02JlXue8SKWv_9X6K_JnnfInPUWqJw</recordid><startdate>20120706</startdate><enddate>20120706</enddate><creator>Herrick-Davis, Katharine</creator><creator>Grinde, Ellinor</creator><creator>Lindsley, Tara</creator><creator>Cowan, Ann</creator><creator>Mazurkiewicz, Joseph E</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20120706</creationdate><title>Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers</title><author>Herrick-Davis, Katharine ; Grinde, Ellinor ; Lindsley, Tara ; Cowan, Ann ; Mazurkiewicz, Joseph E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p266t-4cb77932bf95d2fe36723fd20382076f04be4c859c2ca61e0bbd7c8056316a973</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animals</topic><topic>Cell Membrane - drug effects</topic><topic>Cell Membrane - metabolism</topic><topic>Cells, Cultured</topic><topic>Diffusion - drug effects</topic><topic>Endoplasmic Reticulum - metabolism</topic><topic>Fluorescence</topic><topic>Golgi Apparatus - metabolism</topic><topic>Green Fluorescent Proteins - chemistry</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>HEK293 Cells</topic><topic>Hippocampus - cytology</topic><topic>Humans</topic><topic>Luminescent Proteins - chemistry</topic><topic>Luminescent Proteins - genetics</topic><topic>Luminescent Proteins - metabolism</topic><topic>Microscopy, Confocal</topic><topic>Molecular Biophysics</topic><topic>Mutation</topic><topic>Neurons - cytology</topic><topic>Neurons - drug effects</topic><topic>Neurons - metabolism</topic><topic>Protein Multimerization</topic><topic>Protein Transport - drug effects</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Receptor, Serotonin, 5-HT2C - chemistry</topic><topic>Receptor, Serotonin, 5-HT2C - genetics</topic><topic>Receptor, Serotonin, 5-HT2C - metabolism</topic><topic>Serotonin - pharmacology</topic><topic>Spectrometry, Fluorescence - methods</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Herrick-Davis, Katharine</creatorcontrib><creatorcontrib>Grinde, Ellinor</creatorcontrib><creatorcontrib>Lindsley, Tara</creatorcontrib><creatorcontrib>Cowan, Ann</creatorcontrib><creatorcontrib>Mazurkiewicz, Joseph E</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</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>Herrick-Davis, Katharine</au><au>Grinde, Ellinor</au><au>Lindsley, Tara</au><au>Cowan, Ann</au><au>Mazurkiewicz, Joseph E</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2012-07-06</date><risdate>2012</risdate><volume>287</volume><issue>28</issue><spage>23604</spage><epage>23614</epage><pages>23604-23614</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>22593582</pmid><doi>10.1074/jbc.M112.350249</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Cell Membrane - drug effects Cell Membrane - metabolism Cells, Cultured Diffusion - drug effects Endoplasmic Reticulum - metabolism Fluorescence Golgi Apparatus - metabolism Green Fluorescent Proteins - chemistry Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism HEK293 Cells Hippocampus - cytology Humans Luminescent Proteins - chemistry Luminescent Proteins - genetics Luminescent Proteins - metabolism Microscopy, Confocal Molecular Biophysics Mutation Neurons - cytology Neurons - drug effects Neurons - metabolism Protein Multimerization Protein Transport - drug effects Rats Rats, Sprague-Dawley Receptor, Serotonin, 5-HT2C - chemistry Receptor, Serotonin, 5-HT2C - genetics Receptor, Serotonin, 5-HT2C - metabolism Serotonin - pharmacology Spectrometry, Fluorescence - methods Transfection |
| title | Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers |
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