α(1)‐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 α(1)‐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 α(1)‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α(1)‐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 α(1A/B/D)‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α(1B/D)‐AR:ACKR3, CXCR4:ACKR3, and α(1B/D)‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α(1B/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, α(1A/B/D)‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α(1B/D)‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α(1A/B)‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α(1b)‐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: α(1)‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α(1B/D)‐AR function, and activation of ACKR3 negatively regulates α(1)‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α(1)‐ARs function within a network of hetero‐oligomeric receptor complexes.