Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein

J Mol Biol. 2003 Jun 13;329(4):815-29. doi: 10.1016/s0022-2836(03)00439-x.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Circular Dichroism
  • Cross-Linking Reagents
  • Cysteine / metabolism
  • Dimerization
  • 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 / 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


  • Cross-Linking Reagents
  • Ligands
  • Receptors, Leukotriene B4
  • Recombinant Proteins
  • Guanosine Diphosphate
  • Leukotriene B4
  • Guanosine Triphosphate
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • Heterotrimeric GTP-Binding Proteins
  • Cysteine