α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

BACKGROUND Recently, we provided evidence that α1-adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1-ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and α1-ARs is unknown. METHODS AND RESULTS We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co-immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D-AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D-AR:ACKR3, CXCR4:ACKR3, and α1B/D-AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D-AR:ACKR3 heteromers. Phenylephrine-induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D-AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D-AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B-AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b-AR in β-arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine-induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine-induced constriction of mesenteric arteries. CONCLUSIONS α1-ARs form hetero-oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D-AR function, and activation of ACKR3 negatively regulates α1-ARs. G protein-coupled receptor hetero-oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1-ARs function within a network of hetero-oligomeric receptor complexes.