Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B4 Receptor BLT1 and the G-protein

doi:10.1016/S0022-2836(03)00439-X

Journal of Molecular Biology

Volume 329, Issue 4, 13 June 2003, Pages 815-829

https://doi.org/10.1016/S0022-2836(03)00439-X Get rights and content

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 (K_a=7.8×10⁸ M⁻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 (K_a=1.3×10¹⁰ M⁻¹) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Gα_i2β₁γ₂. 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:Gα_i2β₁γ₂ pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB₄-induced signal.

Introduction

G-protein coupled receptors (GPCRs) are versatile biological sensors that are responsible for the majority of cellular responses to hormones and neurotransmitters as well as for the senses of sight, smell and taste. A growing body of functional and biochemical evidence suggests that GPCRs exist as homo- or heterodimers.1., 2. The majority of studies have relied largely on co-immunoprecipitation and function-restoring approaches. Recently, more direct evidence that GPCRs form dimers has been provided using bioluminescence or fluorescence resonance energy transfer experiments.3., 4., 5. However, there is still little direct information on the GPCR dimerization process. This is largely due to the fact that, with the exception of rhodopsin, the low natural abundance of GPCRs precludes the use of biophysical and biochemical approaches, and that no expression system has been satisfactorily established to produce these receptors, in a functional state, in high yields.

We have produced the leukotriene B₄ receptor BLT1 in Escherichia coli as a functionally and structurally defined protein, isolated in a detergent-containing medium, in yields compatible with the first structural characterization in solution (J.-L.B. et al., accompanying paper⁶). We have shown that recombinant BLT1 binds LTB₄ with an affinity constant, K_a ca 8×10⁸ M⁻¹, and a stoichiometric ratio of one agonist molecule per receptor molecule. This affinity constant is significantly lower than that measured under conditions with human BLT1 expressed in Cos-7 cells (K_a ca 10¹⁰ M⁻¹).⁷ In this work, we investigate both the propensity of BLT1 to form oligomers and its ability to interact with a heterotrimeric G-protein. We report evidence that: (i) upon binding, LTB₄ induces BLT1 dimerization; (ii) receptor dimerization is required for interacting with the heterotrimeric G-protein to form a well-defined pentameric protein assembly with one G-protein and one receptor dimer; (iii) an increase of about tenfold in affinity of the receptor for its agonist occurs upon formation of the pentameric complex; and (iv) GDP-GTP exchange occurs at the level of the pentameric complex and is followed by Gα activation.

Section snippets

G-protein effects on the affinity of recombinant BLT1

In our previous study (J.-L.B et al., accompanying paper⁶), it is shown that detergent-solubilized BLT1 displays an affinity constant for LTB₄ by one order of magnitude lower than that measured in vivo. In light of the observation that high-affinity binding of LTB₄ by BLT1 is apparently dependent on the presence of a G-protein,⁸ we attempted to induce such a high-affinity binding state in our isolated recombinant BLT1 by complementing it with a purified heterotrimeric G-protein. We thus

Discussion

It has been shown, in the case of porcine BLT1 (as part of an isolated membrane fraction), that the K_a value for LTB₄ binding is enhanced by as much as one order of magnitude upon addition of a G-protein,⁸ thus suggesting that association of G with the LTB₄ receptor might affect the affinity for the agonist. We show here with the isolated recombinant human receptor in a detergent environment, that high-affinity LTB₄ binding is indeed observed in the presence of Gα_i2β₁γ₂. The thermodynamic and

Materials and buffers

The antagonist molecule 5bα was synthesized as described.⁹ [³⁵S]GTPγS was from Amersham; 5-hydroxy-d-tryptophan was from Aldrich. All other reagents were as described.⁶ dTSP was from Pierce chemicals. Buffer A was 25 mM potassium phosphate (pH 6.0), 50 mM KCl, 2 mM LDAO. Buffer B was 10 mM sodium borate (pH 8), 50 mM KCl, 2 mM LDAO.

Recombinant proteins

Production of full-length recombinant BLT1 from human origin was carried out in E. coli as described.⁶ Here, we describe in some detail the production of the

Supplementary Files

Acknowledgements

We thank J. Zaccaı̈ for his assistance in the SANS measurements and I.L.L. (Grenoble) for access to the D22 station. We thank CNRS (Programme “Physique-Chimie du Vivant”) and Ministère de la Recherche (Action Concertée Incitative “Molécules et Cibles Thérapeutiques”) for their financial support.

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Journal of Molecular Biology

Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B4 Receptor BLT1 and the G-protein

Abstract

Introduction

Section snippets

G-protein effects on the affinity of recombinant BLT1

Discussion

Materials and buffers

Recombinant proteins

Supplementary Files

Acknowledgements

J. Biol. Chem.

Curr. Biol.

Biophys. J.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Pharmacol. Ther.

J. Biol. Chem.

J. Chromatog. sect. A

Anal. Biochem.

Oligomerization of G-protein-coupled transmitter receptors

Nature Rev. Neurosci.

Oligomerisation of G-protein-coupled receptors

J. Cell Sci.

Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

Proc. Natl Acad. Sci. USA

A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis

Nature

Characterization of the leukotriene B4 receptor in porcine leucocytes

Eur. J. Biochem.

Structure-based Analysis of GPCR Function: Evidence for a Novel Pentameric Assembly between the Dimeric Leukotriene B₄ Receptor BLT1 and the G-protein

Detection of β₂-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

A G-protein-coupled receptor for leukotriene B₄ that mediates chemotaxis

Characterization of the leukotriene B₄ receptor in porcine leucocytes