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 alpha(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 alpha(1)ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and a1-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 alpha(1A/B/D)-AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced alpha(1B/D)-AR: ACKR3, CXCR4: ACKR3, and alpha(1B/D)-AR: CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced alpha(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, alpha(1A/B/D)-AR, and CXCR4. While the transmembrane domain 2 peptide interfered with alpha(1B/D)-AR: ACKR3 and CXCR4: ACKR3 heteromerization, it increased heteromerization between CXCR4 and alpha(1A/B)-AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect alpha(1b)-AR in beta-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-alpha(1)-ARs form hetero-oligomeric complexes with the ACKR3: CXCR4 heteromer, which is required for alpha(1B/D)-AR function, and activation of ACKR3 negatively regulates alpha(1)-ARs. G protein-coupled receptor hetero-oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous a1-ARs function within a network of hetero-oligomeric receptor complexes.