Abstract
Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α1A/B-AR and CXCR4, and inhibited Ca(2+) mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α1-AR activation. CXCR4 silencing reduced α1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca(2+) fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of α1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.
Highlights
Chemokine (C-X-C motif) receptor 4 (CXCR4) is a G proteincoupled receptor (GPCR) that is essential during development
We show that disruption of α1A/B-adrenergic receptors (ARs): CXCR4 heteromeric complexes inhibits α1-AR–mediated functions in vascular smooth muscle cells and that treatment with CXCR4 agonists enhances the potency of the α1-AR agonist phenylephrine to increase blood pressure
We show that disruption of the quaternary structure of the heteromeric receptor complex by targeting transmembrane helix (TM) 2 of CXCR4 and depletion of heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR agonist-induced key signaling events and contraction of vascular smooth muscle cells (VSMC)
Summary
Chemokine (C-X-C motif) receptor 4 (CXCR4) is a G proteincoupled receptor (GPCR) that is essential during development. We provide evidence that α1A/B-adrenergic receptors (AR) form heteromeric complexes with chemokine (C-X-C motif) receptor 4 (CXCR4) on the cell surface of vascular smooth muscle cells. We show that disruption of α1A/B-AR: CXCR4 heteromeric complexes inhibits α1-AR–mediated functions in vascular smooth muscle cells and that treatment with CXCR4 agonists enhances the potency of the α1-AR agonist phenylephrine to increase blood pressure. These findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of G protein-coupled receptor heteromerization with important functional implications. Our observations suggest that α1-AR function in VSMC is controlled through the formation of heteromeric α1A/B-AR:CXCR4 complexes
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