Class A G protein-coupled receptors assemble into functional higher-order hetero-oligomers

Abstract

Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, α_1a -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.

Keywords: ACKR3; AVPR1A; CXCR4; receptor dimer; receptor hetero-oligomerization; ɑ1-adrenergic receptor.

Figure 1.

Heterodimerization between recombinant CXCR4, ACKR3, AVPR1A and α₁-ARs. (a) BRET indicates that AVPR1A interacts with α_1a/b/d-ARs. HEK293T cells were transfected with a fixed amount of AVPR1A-RLuc and increasing amounts of α_1a/b/d-AR-YFP or YFP. 48 h after transfection, YFP fluorescence and luminescence were read as described in Methods. Net BRET (528nm/460nm) was plotted against YFP/luminescence (YFP/Lum). The graph is representative of at least three independent experiments. (b-k) BiLC or BiFC indicates heterodimerization between recombinant CXCR4, ACKR3, AVPR1A and α₁-ARs. HEK293T cells were transfected in triplicate with a pair of L1 and L2 tagged receptors for BiLC or with a pair of V1 and V2 tagged receptors for BiFC at the amounts indicated. mGlu₁R-L1 or mGlu₁R-V2 were used as negative controls. RLU: relative luminescence units. RFU: relative fluorescence units. Figures are representative of three independent experiments for each receptor pair.

Figure 2.

CXCR4 homo-oligomerization and hetero-trimerization. (a/b) BiLC (A) and BiFC (B) indicates that CXCR4 forms homodimers. CXCR4-L1 and -L2 or CXCR4-V1 and –V2 were co-transfected at the amounts indicated. (c) BiFC BRET suggests that CXCR4 forms a homotrimer. Cells were transfected with CXCR4-RLuc or mGlu₁R-Rluc at a fixed amount, and with increasing amounts of CXCR4-V1/-V2. (d) CXCR4 forms a homo-tetramer. Cells were transfected with CXCR4-L1/-L2 or mGlu₁R-L1/-L2 at a fixed amount and with increasing amounts of CXCR4-V1/-V2. (e) The CXCR4 homodimer forms a heterotrimer with AVPR1A. Cells were transfected with CXCR4-L1/-L2 at a fixed amount and with increasing amounts of AVPR1A-YFP or mGlu₁R-YFP. (f) The CXCR4 homodimer forms a heterotrimer with ACKR3. Cells were transfected with ACKR3-RLuc or mGlu₁R-RLuc at a fixed amount and with increasing amounts of CXCR4-V1/-V2. BRET assays were performed as described in Methods. Net BRET was plotted against YFP fluorescence/luminescence (YFP/Lum). Figures are representative of three independent experiments for each condition.

Figure 3.

Formation of hetero-oligomers comprised of CXCR4, ACKR3, α_1a-AR and AVPR1A.(a-c) BiLC and BiFC BRET suggests hetero-trimerization and hetero-tetramerization. HEK293T cells were co-transfected with a fixed amount of AVPR1A-L1/ACKR3-L2 and increasing amounts of α_1a-AR-YFP (a) or CXCR4-YFP (b) or CXCR4-V1/α_1a-AR-V2 (c). BRET assays were performed as described in Methods. Net BRET was plotted against YFP fluorescence/total luminescence (YFP/Lum). Figures are representative of three independent experiments. mGlu₁R was used as a negative control. (d-f) Effects of agonists on BRET of the hetero-tetrameric receptor cluster. Cells were co-transfected with 0.6 μg of AVPR1A-L1/ACKR3-L2 and 1.8 μg of CXCR4-V1/α_1a-AR-V2. 48 h after transfection, cells were treated with different amounts of arginine vasopressin (aVP), CXCL12 or phenylephrine (PE) for 5 min before measuring BRET. The results shown are mean of BRET changes induced by agonists from three independent experiments.

Figure 4.

Agonist-induced coupling of CXCR4 to signaling transducers is modulated by the presence of the heteromerization partners. (a) Quantification of receptor expression levels by flow cytometry in HTLA cells transfected with CXCR4-Tango alone (blue line) or with CXCR4-Tango plus α_1a-AR, AVPR1A and ACKR3 (orange lines). Grey areas: unstained cells. (b) BRET of CXCL12-induced engagement of CXCR4 with Gαi. Cells were co-transfected with CXCR4-RlucII, Gαi-91Venus plus pcDNA3 (control) or receptors as indicated. N=4. BRETnet: BRET ratios at various concentrations of the agonist subtracted by the ratios in the absence of agonist. (c) PRESTO-Tango assay to measure CXCL12-induced recruitment of β-arrestin to CXCR4. Cells were transfected with CXCR4-Tango plus pcDNA3 (control) or receptors as indicated. N=4. */#: p<0.05 vs. control. RLU (fold increase): Relative luminescence units (RLU) measured after stimulation with CXCL12 / RLU of unstimulated cells. (d) Simplified schematic summarizing the regulation of CXCL12-induced CXCR4 coupling to its signaling transducers within hetero-oligomeric receptor complexes. The lengths of the red arrows represent the general tendency of the changes in potency and/or efficacy of CXCL12 to induce coupling of CXCR4 to Gαi or β_arrestin (β_AR).

Figure 5.

Ligand-binding to heteromerization partners regulates agonist-induced coupling of CXCR4 to signaling transducers. BRET of CXCL12-induced engagement of CXCR4 with Gαi was measured in cells co-transfected with CXCR4-RlucII, Gαi-91Venus plus pcDNA3 (control) (a/e) or all heteromerization partners (b/f). PRESTO-Tango assays to measure CXCL12-induced recruitment of β-arrestin to CXCR4 were performed in cells transfected with CXCR4-Tango plus pcDNA3 (control) (c/g) or all heteromerization partners (d/h). Cells were co-stimulated with CXCL12 plus vehicle, phenylephrine (PE, 1 μM) or arginine vasopressin (aVP, 0.1 μM) (a-d) or pre-treated with vehicle, phentolamine (1 μM) or conivaptan (0.1 μM) for 15 min prior to CXCL12 stimulation (e-h). RLU (%): Relative luminescence units in % of the maximal RLU of cells treated with vehicle and CXCL12 (=100%). N=4 per condition. *: p<0.05 for control vs. phenylephrine or phentolamine. #: p<0.05 for control vs. aVP or conivaptan. (i) Simplified schematic summarizing the effects of ligand-binding to heteromerization partners on agonist-induced coupling of CXCR4 to signaling transducers. The lengths of the red arrows represent the general tendency of the changes in potency and/or efficacy of CXCL12 to induce coupling of CXCR4 to Gαi or β_arrestin (β_AR), as compared with cells stimulated with CXCL12 alone (center).

Figure 6.

The regulation of agonist-induced coupling of CXCR4 to signaling transducers by antagonist-binding to α_1a-AR depends on the composition of heteromerization partners and antagonist-binding to heteromerization partners regulates agonist-induced β-arrestin recruitment to AVPR1A. (a-d) BRET of CXCL12-induced engagement of CXCR4 with Gαi was measured in cells co-transfected with CXCR4-RlucII, G αi-91Venus plus receptors as indicated. PRESTO-Tango assays to measure CXCL12-induced recruitment of β-arrestin to CXCR4 were performed in cells transfected with CXCR4-Tango plus receptors as indicated (e-h). Cells were pre-treated with vehicle or phentolamine (1 μM) for 15 min prior to CXCL12 stimulation. N=4 per condition. *: p<0.05. (i) Simplified schematic summarizing the effects of of ACKR3 on CXCL12-induced CXCR4 activation in the presence of antagonist-bound α_1a-AR. The lengths of the red arrows represent the general tendency of the changes in potency and/or efficacy of CXCL12 to induce coupling of CXCR4 to Gαi or β_arrestin (β_AR), as compared with cells stimulated with CXCL12 alone (center). The presence and absence of ACKR3 resulted in the same effects when cells co-expressed CXCR4, α_1a-AR and AVPR1A (not depicted). (j/k) PRESTO-Tango assays to measure aVP-induced recruitment of β-arrestin to AVPR1A were performed in cells transfected with AVPR1A-Tango plus pcDNA3 (control, (i)) or all heteromerization partners (j). Cells were pre-treated with vehicle, phentolamine ((1 μM) or AMD3100 (1 μM) for 15 min prior to aVP stimulation. RLU (%): Relative luminescence units in % of the maximal RLU of cells treated with vehicle and CXCL12 or aVP (=100%). N=4 per condition. *: p<0.05 vehicle vs. phentolamine. #: p<0.05 vehicle vs. AMD3100.