G protein activation by the leukotriene B4 receptor dimer. Evidence for an absence of trans-activation

Abstract

There is compelling evidence that G protein-coupled receptors exist as homo- and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B(4) receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B(4) but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins.

FIGURE 1.

Ligand binding to BLT1_chim. Shown are direct binding of the ALXR ¹²⁵I-labeled WKYMVm agonist (A) and ¹²⁵I-labeled WKYMVm displacement by LXA₄ (closed circles) or LTB₄ (open circles) (B). Ligand-binding experiments were carried out by equilibrium dialysis as described under “Experimental Procedures.” The direct binding data are presented as a plot of the binding degree X as a function of the ligand concentration. The binding degree is defined by bound mol of ligand/mol of receptor. The experiments illustrated here are representative of three independent trials, each performed in duplicate.

FIGURE 2.

Agonist-induced receptor activation. Shown are fluorescence emission spectra of 5HW-labeled wild-type (A) or chimeric (B) receptors in the absence of ligand (free) or in the presence of LTB₄ or LXA₄. Fluorescence emission spectra were recorded as described under “Experimental Procedures.”

FIGURE 3.

Receptor-catalyzed [³⁵S]GTPγS binding to the G protein. GDP/GTP exchange on Gα_i catalyzed by BLT1 or BLT1_chim in the presence of saturating concentrations in LTB₄ or LXA₄, respectively. Data are expressed as the percentage of maximal binding. In all cases, data represent the mean ± S.E. from three independent experiments.

FIGURE 4.

Ligand binding to the BLT1-BLT1_chim heterodimer. Ligand-binding experiments were carried out by equilibrium dialysis as described under “Experimental Procedures” using ³H-labeled LTB (A) or ¹²⁵I-labeled WKYMVm (B). The binding experiments were carried out in the presence (open symbols) or absence (closed symbols) of 500 μm WKYMVm (A) or LTB₄ (B). The binding data are presented as a plot of the binding degree X as a function of the ligand concentration. The binding degree is defined by bound mol of ligand/mol of receptor. The experiments illustrated here are representative of three independent trials, each performed in duplicate.

FIGURE 5.

BLT1-BLT1_chim-catalyzed [³⁵S]GTPγS binding to the G protein. GDP/GTP exchange on Gα_i catalyzed by the BLT1-BLT1_chim heterodimer in the absence of agonist, in the presence of saturating concentrations in LTB₄ or in the presence of saturating concentrations in LXA₄. Data are expressed as the percentage of maximal binding. In all cases, data represent the mean S.E. from three independent experiments.

FIGURE 6.

Receptor activation in the BLT1-BLT1_chim heterodimer. A, relative change in 5HW fluorescence in the BLT1-BLT1_chim dimer where either of the protomers is labeled with 5HW in the absence of agonist or in the presence of either LTB₄ or LXA₄. The species analyzed are schematically depicted in each case, where the open box represents the wild-type protomer, the gray box represents the BLT1chim protomer, and the star represents 5HW labeling. B, relative change in 5HW fluorescence in the BLT1-BLT1_chim dimer, where the labeled protomer is the chimeric one in the absence of ligand, after the addition of LTB₄, or after the addition of LTB₄ and then LXA₄. In all of the cases, the error bar corresponds to the S.D. value calculated from three independent experiments.

FIGURE 7.

Cross-conformational changes in the R-R_chim heterodimer. Fluorescence emission changes of 5HW-labeled R (profiles 1 and 3) or R_chim (profiles 2 and 4) as a function of time. LTB₄ was first bound to the R protomer in the R-R_chim dimer, a large excess in LXA₄ was added (LXA₄/LTB₄ ratio 1000:1), and the changes in the emission properties were recorded as a function of time. Measurements were carried out in the absence (profiles 1 and 2) or the presence (profiles 3 and 4) of 0.1 mm GTP.

FIGURE 8.

BLT1_i3-1-BLT1_chim-catalyzed [³⁵S]GTPγS binding to the G protein. A, GDP/GTP exchange on Gα_i catalyzed by BLT1 or BLT1_i3-1 in the presence of saturating concentrations in LTB₄. Data are expressed as the percentage of maximal binding. The species analyzed are schematically depicted in each case, where the open box represents the wild-type protomer and the cross represents the i3-1 mutation. In all cases, data represent the mean S.E. from three independent experiments. B, GDP/GTP exchange on Gα_i (open boxes) or Gα_o (closed boxes) catalyzed by the BLT1_i3-1-BLT1_chim heterodimer in the absence of agonist, in the presence of saturating concentrations in LTB₄ or in the presence of saturating concentrations in LXA₄. Data are expressed as the percentage of maximal binding. The species analyzed are schematically depicted in each case where the open box represents the wild-type protomer, the gray box represents the BLT1_chim protomer, the cross represents the i3-1 mutation, and the black box represents the ligand. In all cases, data represent the mean S.E. from three independent experiments.

FIGURE 9.

A model for BLT1-BLT1_chim-induced G protein activation. The white box corresponds to BLT1, and the gray box corresponds to BLT1_chim. The G protein is represented by the box marked G. The different shapes correspond to the different conformations adopted by the receptor. Binding of the agonist to one of the protomers (e.g. BLT1) induces a conformational change of the ligand-loaded subunit and a cross-conformational change of the ligand-free protomer. However, only the agonist-loaded protomer can trigger G protein activation.