The chemokine CXC4 and CC2 receptors form homo‐ and heterooligomers that can engage their signaling G‐protein effectors and βarrestin

Abstract

G-protein-coupled receptors have beenshown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo-and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo-and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC 50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of G␣ i1 , G␣ 13 , G␣ z , and ␤arrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents G i activation, also affects the CCR2-promoted engagement of G␣ i1 and ␤arrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo-and heteromultimeric CXCR4 and CCR2 can form functional signaling com- In recent years, biochemical and biophysical studies carried out with purified receptors (1) in heterologous expression systems (2, 3) or native tissue (4 -6) unequivocally demonstrate that many G-protein-coupled receptors (GPCRs) can form homo-and heterooligomers. It was proposed that oligomerization occurs early in the biosynthetic pathway, most likely in the endoplasmic reticulum (ER) where it could play a role in the quality control (7) as well as in anterograde and retrograde trafficking of the receptors to and from the plasma membrane (8). Heterooligomerization has also been proposed to contribute to the pharmacological and signaling diversity. Indeed, it has been shown for many pairs of receptors that the formation of such heterocomplexes can result in binding and/or signaling profiles that differ from those of the homooligomer 1 These authors contributed equally to this work. 2 Current address: S.A., Conseil National de Recherches Canada (CNRC), Édifice Montréal, Montréal, Québec, Canada; J.Q., INSERM, U1016, Institut Cochin, Paris, France; Y.P., Laboratoire de l'Intégration du Matériau au Système (IMS)/CNRS UMR 5218; Université de Bordeaux I, Talence, France. Abbreviations: BiFC, bimolecular fluorescence complementation; BiLC, bimolecular luminescence complementation; BRET, bioluminescence resonance energy transfer; CCR2, chemokine receptor 2; CXCR4, C-X-C chemokine receptor type 4; D2R, D2-dopamine receptor; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; GPCR, G-proteincoupled receptor; MCP-1, monocyte chemotactic protein 1; mGluR, me...

G Protein–Coupled Receptor Multimers: A Question Still Open Despite the Use of Novel Approaches

“…was unable to crossantagonize CCL2-induced b-arrestin2 recruitment to CCR2-CXCR4 heteromers in a CODA-RET assay (Armando et al, 2014). Cannabinoid CB 1 and CB 2 receptor antagonists inhibited signaling in response to agonists that activate the opposite receptor within cannabinoid CB 1 /CB 2 heteromers in both heterologous cells and globus pallidus slices from a rat brain (Callén et al, 2012).…”

“…In addition, fusion of GPCRs to bimolecular luminescence or fluorescence complementation protein fragments and subsequent coexpression with compatible G protein or b-arrestin RET fusion constructs (i.e., CODA-RET) allowed simultaneous detection of GPCR heteromerization and engagement of intracellular signaling partners upon agonist stimulation (Fig. 1H) Armando et al, 2014;Guitart et al, 2014;Bellot et al, 2015;Frederick et al, 2015). Heteromerization of G s -coupled D 1 R and the G i/o -coupled dopamine D 2 receptor (D 2 R) induces intracellular Ca 21 mobilization upon agonist activation, which could be impaired by the G q/11 inhibitor YM254890 Rashid et al, 2007).…”

“…Saturation FRET between purified b 2 -adrenergic receptors (b 2 -ARs) site-specifically labeled with fluorophores and reconstituted in lipid bilayers suggested the predominant formation of tetramers (Fung et al, 2009). Threecolor sequential BRET-FRET and bimolecular luminescence/ fluorescence complementation in combination with resonance energy transfer (RET) revealed the formation of GPCR heteromultimers, consisting of at least three or four individual GPCRs, when expressed at physiologic levels (Lopez-Gimenez et al, 2007;Carriba et al, 2008;Guo et al, 2008;Nijmeijer et al, 2010;Armando et al, 2014). However, the interpretation of quantitative RET approaches between membrane-associated proteins has been challenged (James et al, 2006;Lambert and Javitch, 2014;Lan et al, 2015), and revised experimental designs (Szalai et al, 2014) as well as third-party RET approaches (Kuravi et al, 2010) have been proposed to improve the interpretation of results from RET experiments.…”

Pilot the pulse: controlling the multiplicity of receptor dynamics

“…It is likely that dimerization or oligomerization results in trans-protomer regulation, in other words binding of an agonist to one protomer modulates the signaling output of another protomer, or vice versa, for example as recently shown for a chemokine CXCR4-CCR2 heterotetramer [74][75][76][77][78]. Moreover, dimerization can also have an impact on the signaling network initiated upon receptor activation.…”

FFAR2‐FFAR3 receptor heteromerization modulates short‐chain fatty acid sensing

“…As exemplified by the heteromer of the D 1 -D 2 dopamine (21, 22), GPCRs may interact to form heteromers that display signaling that is distinct from the parent homomers (23–25). Such GPCR heteromers are potential drug targets.…”