Search

It is unknown whether heteromerization between chemokine (C‐X‐C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3) and α1b‐adrenoceptor (α1b‐AR) influences effects of the CXCR4/ACKR3 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) and the noncognate CXCR4 agonist ubiquitin on agonist‐promoted G protein activation. We provide biophysical evidence that both ligands stimulate CXCR4‐mediated Gαi activation. Unlike CXCL12, ubiquitin fails to recruit β‐arrestin. Both ligands differentially modulate the conformation of CXCR4:ACKR3 heterodimers and its propensity to hetero‐trimerize with α1b‐AR. CXCR4:ACKR3 heterodimerization reduces the potency of CXCL12, but not of ubiquitin, to activate Gαi. Ubiquitin enhances phenylephrine‐stimulated α1b‐AR‐promoted Gαq activation from hetero‐oligomers comprising CXCR4. CXCL12 enhances phenylephrine‐stimulated α1b‐AR‐promoted Gαq activation from CXCR4:α1b‐AR heterodimers and reduces phenylephrine‐stimulated α1b‐AR‐promoted Gαq activation from ACKR3 comprising heterodimers and trimers. Our findings suggest heteromer and ligand‐dependent functions of the receptor partners. [ABSTRACT FROM AUTHOR]

Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3–68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05–0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03–3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3–68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3–68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3–68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation. [ABSTRACT FROM AUTHOR]

- Ethanol enhances the propensity of PAR1 and CXCR4 to form heteromers. - Ethanol increases PAR1:CXCR4 heteromer expression in human lung microvascular endothelial cells (HULEC-5a). - Ethanol enhances the efficacy of PAR1 to activate Gα 12 upon thrombin stimulation in cells co-expressing CXCR4. - Ethanol dose-dependently increases the efficacy of thrombin to impair HULEC-5a barrier function at clinically relevant concentrations. - Interference with PAR1:CXCR4 heteromerization mitigates effects of ethanol on thrombin-induced impairment of HULEC-5a barrier function. - Our findings provide a molecular mechanism that is likely to contribute to the increased risk of acute respiratory distress syndrome with alcohol abuse. [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]

We reported previously that α 1 -adrenoceptor (α 1 -AR) ligands inhibit chemokine receptor (CR) heteromerization partners of α 1B/D -AR. The underlying mechanisms are unknown and in vivo evidence for such effects is missing. Utilizing CCR2 and α 1B -AR as prototypical partners, we observed in recombinant systems and THP-1 cells that α 1B -AR enhanced whereas its absence inhibited Gαi signaling of CCR2. Phenylephrine and phentolamine reduced the CCR2:α 1B -AR heteromerization propensity and inhibited Gαi signaling of CCR2. Phenylephrine cross-recruited β-arrestin-2 to CCR2, and reduced expression of α 1B/D -AR, CR partners (CCR1/2, CXCR4) and corresponding heteromers. Phentolamine reduced CR:α 1B/D -AR heteromers without affecting β-arrestin-2 recruitment or receptor expression. Phenylephrine/phentolamine prevented leukocyte infiltration mediated via CR heteromerization partners in a murine air pouch model. Our findings document that α 1 -AR ligands inhibit leukocyte migration mediated by CR heteromerization partners in vivo and suggest interference with α 1B -AR:CR heteromerization as a mechanism by which CR partners are inhibited. These findings provide new insights into the pharmacology of GPCR heteromers and indicate that an agonist and antagonist at one GPCR can act as antagonists at heteromerization partners of their target receptors. [Display omitted] [ABSTRACT FROM AUTHOR]

Highlights: [•] SDF-1α and ubiquitin induce a uniform pattern of CXCR4 internalization. [•] SDF-1α and ubiquitin activate similar signaling cascades downstream of CXCR4. [•] SDF-1α and ubiquitin show partial synergy to activate CXCR4 signaling. [•] The ligand ratio may contribute to the fine tuning of CXCR4 mediated functions. [ABSTRACT FROM AUTHOR]

Extracellular ubiquitin has recently been described as a CXC chemokine receptor (CXCR) 4 agonist. Studies on the structure–function relationship suggested that the C-terminus of ubiquitin facilitates CXCR4 activation. It remains unknown, however, whether C-terminal processing of ubiquitin could be biologically relevant and whether modifications of the ubiquitin C-terminus can modulate CXCR4 activation. We show that C-terminal truncated ubiquitin antagonizes ubiquitin and stromal cell-derived factor (SDF)-1α induced effects on cell signaling and function. Reduction of cell surface expression of insulin degrading enzyme (IDE), which cleaves the C-terminal di-Gly of ubiquitin, enhances ubiquitin induced reduction of cAMP levels in BV2 and THP-1 cells, but does not influence changes in cAMP levels in response to SDF-1α. Reduction of cell surface IDE expression in THP-1 cells also increases the chemotactic activity of ubiquitin. As compared with native ubiquitin, C-terminal Tyr extension of ubiquitin results in reduced CXCR4 mediated effects on cellular cAMP levels and abolishes chemotactic activity. Replacement of C-terminal di-Gly of ubiquitin with di-Val or di-Arg enhances CXCR4 mediated effects on cAMP levels and the di-Arg substitution exerts increased chemotactic activity, when compared with wild type ubiquitin. The chemotactic activities of the di-Val and di-Arg mutants and their effects on cAMP levels can be antagonized with C-terminal truncated ubiquitin. These data suggest that the development of CXCR4 ligands with enhanced agonist activities is possible and that C-terminal processing of ubiquitin could constitute a biological mechanism, which regulates termination of receptor signaling. [ABSTRACT FROM AUTHOR]

CXC chemokine receptor (CXCR)-4 agonists have been shown to attenuate inflammation and organ injury in various disease models, including trauma/hemorrhage. The pathophysiological role of CXCR4 during the early response to tissue injury, however, remains unknown. Therefore, we investigated the effects of AMD3100, a drug that antagonizes binding of stromal cell-derived factor (SDF)-1α and ubiquitin to CXCR4 during the initial response to polytrauma in pigs. Fifteen minutes before polytrauma (femur fractures/lung contusion; control: sham), 350 nmol/kg AMD3100, equimolar AMD3100 and ubiquitin (350 nmol/kg each) or vehicle were administered intravenously. After a 60-min shock period, fluid resuscitation was performed for 360 min. Ubiquitin binding to peripheral blood mononuclear cells was significantly reduced after intravenous AMD3100. SDF-1α plasma levels increased transiently >10-fold with AMD3100 in all animals. In injured animals, AMD3100 increased fluid requirements to maintain hemodynamics and enhanced increases in peripheral blood granulocytes, lymphocytes and monocytes, compared with its effects in uninjured animals. Cytokine release from leukocytes in response to Toll-like receptor (TLR)-2 and TLR-4 activation was increased after in vitro AMD3100 treatment of normal whole blood and after in vivo AMD3100 administration in animals subjected to polytrauma. Coadministration of AMD3100/ubiquitin reduced lactate levels, prevented AMD3100-induced increases in fluid requirements and sensitization of the tumor necrosis factor (TNF)-α and interleukin (IL)-6 release upon TLR-2/4 activation, but did not attenuate increases in leukocyte counts and SDF-1α plasma levels. Our findings suggest that CXCR4 controls leukocyte mobilization after trauma, regulates leukocyte reactivity toward inflammatory stimuli and mediates protective effects during the early phase of trauma-induced inflammation. [ABSTRACT FROM AUTHOR]