Biochemical and Biophysical Research Communications, Vol.528, No.2, 368-375, 2020
Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and alpha(1)-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays
Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with alpha(1)-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:alpha(1)-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:alpha(1a/b/d)-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGlu(1)R) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or alpha(1a/b/d)-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGlu(1)R-EYFP were nonspecific. BRET50 for CXCR4:ACKR3 and CXCR4:alpha(1a/b/d)-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRETmax of CXCR4: alpha(1a/b/d)-AR heteromers without affecting BRET50; stimulation with CXCL12 reduced BRETmax of CXCR4: alpha(1a-)AR heteromers, but did not affect BRET50 or BRETmax/50 for CXCR4:alpha(1b/d)-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRETmax of CXCR4:alpha(1a/b/d)-AR heteromers and increased BRET(5)0 of CXCR4:alpha(1a/b)-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, a1a-AR and mGlu1R homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric alpha(1a)-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGlu(1)R was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and alpha(1)-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 alpha(1)-ARs constitutively form higher-order hetero-oligomeric receptor clusters. (C) 2020 Elsevier Inc. All rights reserved.