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

BackgroundRecently, 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 ResultsWe 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.Conclusionsα1‐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.

Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

“…Thus, the large differences in the potencies of prazosin and cyclazsosin in the present study could be explained by variations of their potencies for recombinant and endogenous receptors. Moreover, it appears possible that both drugs exert differential pharmacological behaviors upon binding to receptor homomers, which is likely in the expression system, and heteromers in hVSMCs[ 7 – 10 ]. We reported previously that phenylephrine stimulation induces β-arrestin cross-recruitment to and internalization of CXCR4 within the α 1b -AR:CXCR4 heteromer, and that phenylephrine inhibits CXCL12-mediated chemotaxis of hVSMC with high potency and efficacy[ 10 ].…”

“…The assay was performed as recently described [ 7 , 11 – 13 ]. HTLA cells (2.5x10 5 /well) were seeded in a 6-well plate and transfected with 750 ng of each of the TANGO plasmids using Lipofectamine 3000 (ThermoScientific).…”

“…Recently, we showed that α 1 -ARs form hetero-oligomeric complexes with chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 in human vascular smooth muscle cells (hVSMC), through which the chemokine receptors regulate α 1 -AR signaling and function[ 7 – 9 ]. Subsequently, we provided evidence for asymmetrical cross-regulation of CXCR4-mediated signaling and function by α 1 -ARs within the heteromeric receptor complex[ 10 ].…”

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

“…PLA were performed as described in detail previously [ 34 – 36 ], utilizing mouse anti-ACKR3 (R&D MAB42273) and goat anti-CXCR4 (Abcam Ab1670). The antibodies have been validated for their receptor target previously [ 35 – 37 ].…”

Context-Dependent Signaling of CXC Chemokine Receptor 4 and Atypical Chemokine Receptor 3

“…Regarding ACKR3 heteromerization, there is evidence of the presence of a 1 -AR:ACKR3:CXCR4 hetero-oligomers in VSMCs, and the activation of ACKR3 can lead to the inhibition of the a 1 -AR activity (Albee et al, 2017). ACKR3 is also known to interact with the epidermal growth factor receptor (EGFR) in a b-arrestin-2-dependent manner and is implicated in the phosphorylation of the EGFR.…”