Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B4 Receptor BLT1 and the G-protein
We produced human leukotriene B4 (LTB4) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB4: (i) a low-affinity state (Ka=7.8×108M−1) that involves a receptor homodimer (BLT1·LTB4)2; we report eviden...
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| Veröffentlicht in: | Journal of molecular biology 2003-06, Vol.329 (4), p.815-829 |
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| creator | Banères, Jean-Louis Parello, Joseph |
| description | We produced human leukotriene B4 (LTB4) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB4: (i) a low-affinity state (Ka=7.8×108M−1) that involves a receptor homodimer (BLT1·LTB4)2; we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (Ka=1.3×1010M−1) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Gαi2β1γ2. Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Gα subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB4, association with a G-protein and activation of Gα. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1·LTB4)2:Gαi2β1γ2 pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB4-induced signal. |
| doi_str_mv | 10.1016/S0022-2836(03)00439-X |
| format | Article |
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This detergent-solubilized receptor displays two states with regard to its affinity for LTB4: (i) a low-affinity state (Ka=7.8×108M−1) that involves a receptor homodimer (BLT1·LTB4)2; we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (Ka=1.3×1010M−1) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Gαi2β1γ2. Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Gα subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB4, association with a G-protein and activation of Gα. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1·LTB4)2:Gαi2β1γ2 pentameric assembly. 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This detergent-solubilized receptor displays two states with regard to its affinity for LTB4: (i) a low-affinity state (Ka=7.8×108M−1) that involves a receptor homodimer (BLT1·LTB4)2; we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (Ka=1.3×1010M−1) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Gαi2β1γ2. Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Gα subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB4, association with a G-protein and activation of Gα. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1·LTB4)2:Gαi2β1γ2 pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB4-induced signal.</description><subject>BLT1</subject><subject>BLT1:G-protein complex</subject><subject>Circular Dichroism</subject><subject>Cross-Linking Reagents</subject><subject>Cysteine - metabolism</subject><subject>Dimerization</subject><subject>Escherichia coli</subject><subject>Escherichia coli - metabolism</subject><subject>Fluorescence</subject><subject>GTP-Binding Protein alpha Subunits, Gi-Go - chemistry</subject><subject>GTP-Binding Protein alpha Subunits, Gi-Go - metabolism</subject><subject>GTP-Binding Proteins - metabolism</subject><subject>Guanosine Diphosphate - metabolism</subject><subject>Guanosine Triphosphate - metabolism</subject><subject>Heterotrimeric GTP-Binding Proteins - chemistry</subject><subject>Heterotrimeric GTP-Binding Proteins - metabolism</subject><subject>Humans</subject><subject>leukotriene B4</subject><subject>Leukotriene B4 - metabolism</subject><subject>Ligands</subject><subject>Models, Molecular</subject><subject>Neutrons</subject><subject>Protein Conformation</subject><subject>Receptors, Leukotriene B4 - agonists</subject><subject>Receptors, Leukotriene B4 - chemistry</subject><subject>Receptors, Leukotriene B4 - genetics</subject><subject>Receptors, Leukotriene B4 - metabolism</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - metabolism</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><subject>Structure-Activity Relationship</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1u1DAURi0EokPhEUBeIVgE7NiJbTbVdNoOSCOo2iJ1Z8XOjTAkzmA7U82L8Lx4fgTLrrz4zr2fdQ9Cryn5QAmtP94SUpZFKVn9jrD3hHCmivsnaEaJVIWsmXyKZv-QE_Qixp-EkIpx-Ryd0FJIUUsyQ39uU5hsmgIUponQ4rlv-m10EY8dXl4vbvDV5G1yo_-ELzeuBW8Bd2PADf46bqDH1-BTM0BwFs9jhMH0W2wgPQB4nH4AvnCHcAXTrzEFBx7wOcc3YGGd8p7z1R3FjW_38LJYhzGB8y_Rs67pI7w6vqfo-9Xl3eJzsfq2_LKYrwrLBEtFZ2lprBRVqyitSN0qUamcSE4oKF4qaE1VljkyslGVsVVHJTcdrztR1jVhp-jtYW_u_T1BTHpw0ULfNx7GKWrBGOdC8UdBqkSt8g8yWB1AG8YYA3R6HdzQhK2mRO_M6b05vdOiCdN7c_o-z705FkxmgPb_1FFVBs4OAOR7bBwEHa3b6WhdAJt0O7pHKv4Cn6qn0w</recordid><startdate>20030613</startdate><enddate>20030613</enddate><creator>Banères, Jean-Louis</creator><creator>Parello, Joseph</creator><general>Elsevier Ltd</general><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>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20030613</creationdate><title>Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B4 Receptor BLT1 and the G-protein</title><author>Banères, Jean-Louis ; Parello, Joseph</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c373t-fc12bc875d911506d97593738401e9429edb522150b8a95bc5f184bf46f726603</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>BLT1</topic><topic>BLT1:G-protein complex</topic><topic>Circular Dichroism</topic><topic>Cross-Linking Reagents</topic><topic>Cysteine - metabolism</topic><topic>Dimerization</topic><topic>Escherichia coli</topic><topic>Escherichia coli - metabolism</topic><topic>Fluorescence</topic><topic>GTP-Binding Protein alpha Subunits, Gi-Go - chemistry</topic><topic>GTP-Binding Protein alpha Subunits, Gi-Go - metabolism</topic><topic>GTP-Binding Proteins - metabolism</topic><topic>Guanosine Diphosphate - metabolism</topic><topic>Guanosine Triphosphate - metabolism</topic><topic>Heterotrimeric GTP-Binding Proteins - chemistry</topic><topic>Heterotrimeric GTP-Binding Proteins - metabolism</topic><topic>Humans</topic><topic>leukotriene B4</topic><topic>Leukotriene B4 - metabolism</topic><topic>Ligands</topic><topic>Models, Molecular</topic><topic>Neutrons</topic><topic>Protein Conformation</topic><topic>Receptors, Leukotriene B4 - agonists</topic><topic>Receptors, Leukotriene B4 - chemistry</topic><topic>Receptors, Leukotriene B4 - genetics</topic><topic>Receptors, Leukotriene B4 - metabolism</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - metabolism</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Banères, Jean-Louis</creatorcontrib><creatorcontrib>Parello, Joseph</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Banères, Jean-Louis</au><au>Parello, Joseph</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B4 Receptor BLT1 and the G-protein</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>2003-06-13</date><risdate>2003</risdate><volume>329</volume><issue>4</issue><spage>815</spage><epage>829</epage><pages>815-829</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><abstract>We produced human leukotriene B4 (LTB4) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB4: (i) a low-affinity state (Ka=7.8×108M−1) that involves a receptor homodimer (BLT1·LTB4)2; we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (Ka=1.3×1010M−1) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Gαi2β1γ2. Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Gα subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB4, association with a G-protein and activation of Gα. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1·LTB4)2:Gαi2β1γ2 pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB4-induced signal.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>12787680</pmid><doi>10.1016/S0022-2836(03)00439-X</doi><tpages>15</tpages></addata></record> |
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| ispartof | Journal of molecular biology, 2003-06, Vol.329 (4), p.815-829 |
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| subjects | BLT1 BLT1:G-protein complex Circular Dichroism Cross-Linking Reagents Cysteine - metabolism Dimerization Escherichia coli Escherichia coli - metabolism Fluorescence GTP-Binding Protein alpha Subunits, Gi-Go - chemistry GTP-Binding Protein alpha Subunits, Gi-Go - metabolism GTP-Binding Proteins - metabolism Guanosine Diphosphate - metabolism Guanosine Triphosphate - metabolism Heterotrimeric GTP-Binding Proteins - chemistry Heterotrimeric GTP-Binding Proteins - metabolism Humans leukotriene B4 Leukotriene B4 - metabolism Ligands Models, Molecular Neutrons Protein Conformation Receptors, Leukotriene B4 - agonists Receptors, Leukotriene B4 - chemistry Receptors, Leukotriene B4 - genetics Receptors, Leukotriene B4 - metabolism Recombinant Proteins - chemistry Recombinant Proteins - metabolism Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure-Activity Relationship |
| title | Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B4 Receptor BLT1 and the G-protein |
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