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

Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α-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-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGluR) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGluR-EYFP were nonspecific. BRET for CXCR4:ACKR3 and CXCR4:α-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRET of CXCR4:α-AR heteromers without affecting BRET; stimulation with CXCL12 reduced BRET of CXCR4:α-AR heteromers, but did not affect BRET or BRET for CXCR4:α-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRET of CXCR4:α-AR heteromers and increased BRET of CXCR4:α-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α-AR and mGluR homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGluR was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α-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 α-ARs constitutively form higher-order hetero-oligomeric receptor clusters.