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 thatCXCR4 controls α1‐ARsthrough formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk betweenACKR3 and α1‐ARsis unknown. Methods and Results We show thatACKR3 agonists inhibit inositol trisphosphate production inhVSMCson stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenousACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown ofACKR3 inhVSMCsreduced α1B/D‐AR:ACKR3,CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown ofCXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production fromhVSMCswas abolished afterACKR3 andCXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 ofACKR3 showed differential effects on heteromerization betweenACKR3, α1A/B/D‐AR,andCXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 andCXCR4:ACKR3 heteromerization, it increased heteromerization betweenCXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibitedACKR3 but did not affect α1b‐ARin 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‐ARsform hetero‐oligomeric complexes with theACKR3:CXCR4 heteromer, which is required for α1B/D‐ARfunction, and activation ofACKR3 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.