Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α1-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays

Abstract

Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α₁-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α₁-AR heteromers, however, is lacking. Here we utilized bimolecular luminescence/fluorescence complementation (BiLC/BiFC) combined with intermolecular bioluminescence resonance energy transfer (BRET) assays in HEK293T cells to evaluate CXCR4:α_1a/b/d-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGlu₁R) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α_1a/b/d-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGlu₁R-EYFP were nonspecific. BRET₅₀ for CXCR4:ACKR3 and CXCR4:α_1a/b/d-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRET_max of CXCR4:α_1a/b/d-AR heteromers without affecting BRET₅₀; stimulation with CXCL12 reduced BRET_max of CXCR4:α_1a-AR heteromers, but did not affect BRET₅₀ or BRET_max/50 for CXCR4:α_1b/d-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRET_max of CXCR4:α_1a/b/d-AR heteromers and increased BRET₅₀ of CXCR4:α_1a/b-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α_1a-AR and mGlu₁R homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α_1a-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGlu₁R was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α₁-ARs is comparable, provide evidence for conformational changes of the receptor complexes upon agonist binding and support the concept that proto- and oligomeric CXCR4 and α₁-ARs constitutively form higher-order hetero-oligomeric receptor clusters.

Keywords: Bimolecular fluorescence complementation assay; Bimolecular luminescence complementation assay; G protein-coupled receptor heteromers; Hetero-oligomer; Homodimer; Stromal cell-derived factor 1α.

Figure 1:

BRET assays indicate that CXCR4 interacts with α_1a/b/d-ARs. Graphs are representative of at least three independent experiments. A/B. HEK293T cells were transfected with a fixed amount of CXCR4-Rluc and increasing amounts of ACKR3-EYFP, α_1a/b/d-AR-EYFP, EYFP (A) or mGlu1R-EYFP (B). 48 h after transfection, EYFP fluorescence and luminescence were read as described in Methods. Net BRET (528nm/460nm) was plotted against EYFP/luminescence (YFP/Lum). C. HEK293T cells were transfected with increasing amounts of both CXCR4-Rluc and ACKR3-EYFP or :α_1a/b/d-AR-EYFP at a fixed ratio (1:10) in quadruplicate. Raw BRET (528nm/460nm) was plotted against total DNA amounts transfected.

Figure 2:

Effects of phenylephrine and CXCL12 on BRET between CXCR4 and α_1a/b/d-AR. HEK293T cells were co-transfected with a fixed amount of CXCR4-Rluc and increasing amounts α_1a-AR-EYFP (A), α_1b-AR-EYFP (B) or α_1d-AR-EYFP (C). 48 h after transfection, cells were treated with vehicle (=control), phenylephrine (PE, 200 μM) or CXCL12 (500 nM) for 5 min at 37°C before measuring BRET. Top: Representative measurements from a titration BRET experiment. Center: BRET₅₀ from n=4 independent titration BRET experiments. Bottom: BRET_max from n=4 independent titration BRET experiments. *: p<0.05 vs. control.

Figure 3:

A peptide analogue derived from TM2 of CXCR4 interferes with BRET between CXCR4 and α_1a/b/d-ARs. HEK293T cells were co-transfected with a fixed amount of CXCR4-Rluc and increasing amounts of α_1a-AR-EYFP (A), α_1b-AR-EYFP (B) or α_1d-AR-EYFP (C). 48 h after transfection, cells were treated with vehicle (=control), TM2 or TM4 peptides (20 μM) for 15 min at 37°C before measuring BRET. Top: Representative measurements from a titration BRET experiment. Center: BRET₅₀ from n=4 independent titration BRET experiments. *: p<0.05 vs. control. Bottom: BRET_max from n=4 independent titration BRET experiments. *: p<0.05 vs. control.

Figure 4:

The CXCR4 homodimer interacts with α_1a-AR. A/B. Bimolecular luminescence complementation (BiLC, A) and bimolecular fluorescence complementation (BiFC, B) assays to detect dimeric CXCR4. HEK293T cells were co-transfected with CXCR4-L1/2 or CXCR4-V1/2, as indicated. Luminescence was read after the addition of coelenterazine H. N=3. C/D. BiLC and BiFC BRET indicate that the CXCR4 homodimer interacts with α_1a-AR. HEK293T cells were co-transfected with a constant amount of CXCR4-L1/2 and with increasing amounts of α_1a-AR -EYFP or EYFP (control, C), or with a constant amount of α_1a-AR-RLuc or mGlu₁R-RLuc and with increasing amounts of CXCR4-V1/2 (control, D). 48 h after transfection, EYFP fluorescence and luminescence were read as described in the Methods. The net BRET (528/460) is plotted against YFP/Lum. The figure is representative of three independent experiments.

Figure 5:

Homodimeric CXCR4 interacts with homodimeric α_1a-AR. A/B. Bimolecular luminescence complementation (BiLC) assays to detect dimeric mGLu₁R (A) and dimeric α_1a-AR (B). HEK293T cells were co-transfected with mGLu₁R-L1/2 or α_1a-AR-L1/2, as indicated. Luminescence was read after the addition of coelenterazine H. N=3. C. Combined BiLC and BiFC BRET indicates that the CXCR4 homodimer interacts with the α_1a-AR homodimer, but not with the mGlu₁R homodimer. HEK293T cells were co-transfected with constant amounts of α_1a-AR-L1/2 or mGlu₁R-L1/L2 and with increasing amounts of CXCR4-V1/V2. 48 h after transfection, EYFP fluorescence and luminescence were read as described in the Methods. The net BRET (528/460) is plotted against YFP/Lum. The figure is representative of three independent experiments.