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

SUBMITTER: Albee LJ

PROVIDER: S-EPMC5586474 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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α<sub>1</sub>-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.

Albee Lauren J LJ Eby Jonathan M JM Tripathi Abhishek A LaPorte Heather M HM Gao Xianlong X Volkman Brian F BF Gaponenko Vadim V Majetschak Matthias M

Journal of the American Heart Association 20170817 8

<h4>Background</h4>Recently, we provided evidence that α<sub>1</sub>-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 α<sub>1</sub>-ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and α<sub>1</sub>-ARs is unknown.<h4>Methods and results</h4>We sh ...[more]

PMID: 28862946

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Project description:We observed in PRESTO-Tango β-arrestin recruitment assays that the α1-adrenergic receptor (AR) antagonist prazosin activates chemokine (C-X-C motif) receptor (CXCR)4. This prompted us to further examine this unexpected pharmacological behavior. We screened a panel of 14 α1/2- and β1/2/3-AR antagonists for CXCR4 and atypical chemokine receptor (ACKR)3 agonist activity in PRESTO-Tango assays against the cognate agonist CXCL12. We observed that multiple α1-AR antagonists activate CXCR4 (CXCL12 = prazosin = cyclazosin > doxazosin) and ACKR3 (CXCL12 = prazosin = cyclazosin > alfuzosin = doxazosin = phentolamine > terazosin = silodosin = tamsulosin). The two strongest CXCR4/ACKR3 activators, prazosin and cyclazosin, were selected for a more detailed evaluation. We found that the drugs dose-dependently activate both receptors in β-arrestin recruitment assays, stimulate ERK1/2 phosphorylation in HEK293 cells overexpressing each receptor, and that their effects on CXCR4 could be inhibited with AMD3100. Both α1-AR antagonists induced significant chemical shift changes in the 1H-13C-heteronuclear single quantum correlation spectrum of CXCR4 and ACKR3 in membranes, suggesting receptor binding. Furthermore, prazosin and cyclazosin induced internalization of endogenous CXCR4/ACKR3 in human vascular smooth muscle cells (hVSMC). While these drugs did not in induce chemotaxis in hVSMC, they inhibited CXCL12-induced chemotaxis with high efficacy and potency (IC50: prazosin-4.5 nM, cyclazosin 11.6 pM). Our findings reveal unexpected pharmacological properties of prazosin, cyclazosin, and likely other α1-AR antagonists. The results of the present study imply that prazosin and cyclazosin are biased or partial CXCR4/ACKR3 agonists, which function as potent CXCL12 antagonists. Our findings could provide a mechanistic basis for previously observed anti-cancer properties of α1-AR antagonists and support the concept that prazosin could be re-purposed for the treatment of disease processes in which CXCR4 and ACKR3 are thought to play significant pathophysiological roles, such as cancer metastases or various autoimmune pathologies.

Project description:Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α1-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α1-AR heteromers, however, is lacking. Here we utilized bimolecular luminescence/fluorescence complementation (BiLC/BiFC) combined with intermolecular bioluminescence resonance energy transfer (BRET) assays in HEK293T cells to evaluate CXCR4:α1a/b/d-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGlu1R) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α1a/b/d-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGlu1R-EYFP were nonspecific. BRET50 for CXCR4:ACKR3 and CXCR4:α1a/b/d-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRETmax of CXCR4:α1a/b/d-AR heteromers without affecting BRET50; stimulation with CXCL12 reduced BRETmax of CXCR4:α1a-AR heteromers, but did not affect BRET50 or BRETmax/50 for CXCR4:α1b/d-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRETmax of CXCR4:α1a/b/d-AR heteromers and increased BRET50 of CXCR4:α1a/b-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α1a-AR and mGlu1R homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α1a-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGlu1R was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α1-ARs is comparable, provide evidence for conformational changes of the receptor complexes upon agonist binding and support the concept that proto- and oligomeric CXCR4 and α1-ARs constitutively form higher-order hetero-oligomeric receptor clusters.

Dataset Information

?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.

Publications

α<sub>1</sub>-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.

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