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Previously, we reported that chemokine (C‐C motif) receptor 2 (CCR2) heteromerizes with α1B‐adrenoceptor (α1B‐AR) in leukocytes, through which α1B‐AR controls CCR2. Whether such heteromers are expressed in human vascular smooth muscle cells (hVSMCs) is unknown. Bioluminescence resonance energy transfer confirmed formation of recombinant CCR2:α1b‐AR heteromers. Proximity ligation assays detected CCR2:α1B‐AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α1B‐AR heteromerization per se enhanced α1B‐AR‐mediated Gαq‐coupling. Chemokine (C‐C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gαq activation via α1B‐AR, cross‐recruited β‐arrestin to and induced internalization of α1B‐AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α1B‐AR heteromers biases α1B‐AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function. [ABSTRACT FROM AUTHOR]

Chemokine receptor antagonists are being explored for their therapeutic potential in various disease processes. As the chemokine (C–C motif) receptor 2 (CCR2) antagonist RS504393 is known to compete with ligand binding to α1-adrenoceptors, we tested a panel of 10 CCR antagonists for interactions with α1-adrenoceptors to evaluate potential cardiovascular activities and side-effect profiles. The PRESTO-Tango β-arrestin recruitment assay was utilized to test whether the CCR antagonists interfere with α1b-AR activation upon stimulation with phenylephrine. Pressure myography with isolated rat resistance arteries was employed to assess their effects on phenylephrine-induced vasoconstriction. The following antagonists were tested: CCR1–BX471, BX513, BI639667; CCR2–RS504393, INCB3284; CCR3–SB328437; and CCR4–AZD2098, and C021; CCR5–Maraviroc; CCR10-BI6901. The pan-α1-adrenoceptor antagonist prazosin was used as control. Among the CCR antagonists tested, RS504393, BX513, and C021 inhibited phenylephrine-induced β-arrestin recruitment to α1b-adrenoceptor and phenylephrine-induced vasoconstriction. While RS504393 functioned as a competitive α1-adrenoceptor blocker, BX513 and C021 functioned as noncompetitive α1-adrenoceptor antagonists in both assay systems. Furthermore, RS504393, BX513, and C021 dose-dependently dilated arteries that were fully preconstricted with phenylephrine. Our data suggest that CCR antagonists should be screened for cross-reactivity with α1-adrenoceptors to exclude potential adverse cardiovascular effects when used as anti inflammatory drugs. [ABSTRACT FROM AUTHOR]

Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α 1 -adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α 1 -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:α 1a/b/d -AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGlu 1 R) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α 1a/b/d -ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGlu 1 R-EYFP were nonspecific. BRET 50 for CXCR4:ACKR3 and CXCR4:α 1a/b/d -AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRET max of CXCR4:α 1a/b/d -AR heteromers without affecting BRET 50 ; stimulation with CXCL12 reduced BRET max of CXCR4:α 1a -AR heteromers, but did not affect BRET 50 or BRET max/50 for CXCR4:α 1b/d -AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRET max of CXCR4:α 1a/b/d -AR heteromers and increased BRET 50 of CXCR4:α 1a/b -AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α 1a -AR and mGlu 1 R homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α 1a -AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGlu 1 R was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α 1 -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 α 1 -ARs constitutively form higher-order hetero-oligomeric receptor clusters. • Luminescence/fluorescence complementation combined with bioluminescence resonance energy transfer assays were utilized. • Proto- and oligomeric CXCR4 and α 1 -adrenoceptors form higher-order hetero-oligomeric receptor complexes. • The heteromerization affinity of CXCR4 for ACKR3 and α 1 -adrenoceptors is comparable. • CXCR4:α 1 -adrenoceptor hetero-oligomers undergo conformational changes upon agonist binding. [ABSTRACT FROM AUTHOR]

The chemokine receptor CXCR4, a G rotein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)- induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce β-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin- induced b-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (CXC motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption. [ABSTRACT FROM AUTHOR]

Previously, we reported that chemokine (C-C motif) receptor 2 (CCR2) heteromerizes with α 1B -adrenoceptor (α 1B -AR) in leukocytes, through which α 1B -AR controls CCR2. Whether such heteromers are expressed in human vascular smooth muscle cells (hVSMCs) is unknown. Bioluminescence resonance energy transfer confirmed formation of recombinant CCR2:α 1b -AR heteromers. Proximity ligation assays detected CCR2:α 1B -AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α 1B -AR heteromerization per se enhanced α 1B -AR-mediated Gα q -coupling. Chemokine (C-C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gα q activation via α 1B -AR, cross-recruited β-arrestin to and induced internalization of α 1B -AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α 1B -AR heteromers biases α 1B -AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function., (© 2022 Federation of European Biochemical Societies.)

Objectives: Chemokine receptor antagonists are being explored for their therapeutic potential in various disease processes. As the chemokine (C-C motif) receptor 2 (CCR2) antagonist RS504393 is known to compete with ligand binding to α 1 -adrenoceptors, we tested a panel of 10 CCR antagonists for interactions with α 1 -adrenoceptors to evaluate potential cardiovascular activities and side-effect profiles., Methods: The PRESTO-Tango β-arrestin recruitment assay was utilized to test whether the CCR antagonists interfere with α 1b -AR activation upon stimulation with phenylephrine. Pressure myography with isolated rat resistance arteries was employed to assess their effects on phenylephrine-induced vasoconstriction. The following antagonists were tested: CCR1-BX471, BX513, BI639667; CCR2-RS504393, INCB3284; CCR3-SB328437; and CCR4-AZD2098, and C021; CCR5-Maraviroc; CCR10-BI6901. The pan-α 1 -adrenoceptor antagonist prazosin was used as control., Results: Among the CCR antagonists tested, RS504393, BX513, and C021 inhibited phenylephrine-induced β-arrestin recruitment to α 1b -adrenoceptor and phenylephrine-induced vasoconstriction. While RS504393 functioned as a competitive α 1 -adrenoceptor blocker, BX513 and C021 functioned as noncompetitive α 1 -adrenoceptor antagonists in both assay systems. Furthermore, RS504393, BX513, and C021 dose-dependently dilated arteries that were fully preconstricted with phenylephrine., Conclusions: Our data suggest that CCR antagonists should be screened for cross-reactivity with α 1 -adrenoceptors to exclude potential adverse cardiovascular effects when used as anti inflammatory drugs., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)-induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce β-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca 2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin-induced β-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (C X C motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Gao et al.)