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 B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(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 (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha. 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.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.
Banères
Jean-Louis
JL
UMR 5074 CNRS, Chimie Biomoléculaire et Interactions Biologiques, Faculté de Pharmacie, 15 avenue Ch. Flahault, BP 14491, 34093 Cedex 05, Montpellier, France. baneres@pharma.univ-montp1.fr
Parello
Joseph
J
D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007975:Leukotriene B4 / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009502:Neutrons; D011487:Protein Conformation; D018102:Receptors, Leukotriene B4 / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D008024:Ligands; D018102:Receptors, Leukotriene B4; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D007975:Leukotriene B4; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine