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 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.