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

Abstract

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 ( ACKR 3). While we showed that CXCR 4 controls α 1 ‐ ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells ( hVSMCs ), the molecular basis underlying cross‐talk between ACKR 3 and α 1 ‐ ARs is unknown. Methods and Results We show that ACKR 3 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 ACKR 3 form heteromeric complexes with α 1A/B/D ‐ AR . While small interfering RNA knockdown of ACKR 3 in hVSMCs reduced α 1B/D ‐ AR : ACKR 3, CXCR 4: ACKR 3, and α 1B/D ‐ AR : CXCR 4 complexes, small interfering RNA knockdown of CXCR 4 reduced α 1B/D ‐ AR : ACKR 3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR 3 and CXCR 4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR 3 showed differential effects on heteromerization between ACKR 3, α 1A/B/D ‐ AR, and CXCR 4. While the transmembrane domain 2 peptide interfered with α 1B/D ‐ AR : ACKR 3 and CXCR 4: ACKR 3 heteromerization, it increased heteromerization between CXCR 4 and α 1A/B ‐ AR . The transmembrane domain 2 peptide inhibited ACKR 3 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 ACKR 3: CXCR 4 heteromer, which is required for α 1B/D ‐ AR function, and activation of ACKR 3 negatively regulates α 1 ‐ AR s. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α 1 ‐ AR s function within a network of hetero‐oligomeric receptor complexes.