α1B/D-adrenoceptors regulate chemokine receptor-mediated leukocyte migration via formation of heteromeric receptor complexes.

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

α1-adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α1B/D-adrenoceptors via interference with heteromer formation.

We reported previously that α1-adrenoceptor (α1-AR) ligands inhibit chemokine receptor (CR) heteromerization partners of α1B/D-AR. The underlying mechanisms are unknown and in vivo evidence for such effects is missing. Utilizing CCR2 and α1B-AR as prototypical partners, we observed in recombinant systems and THP-1 cells that α1B-AR enhanced whereas its absence inhibited Gαi signaling of CCR2. Phenylephrine and phentolamine reduced the CCR2:α1B-AR heteromerization propensity and inhibited Gαi signaling of CCR2. Phenylephrine cross-recruited ß-arrestin-2 to CCR2, and reduced expression of α1B/D-AR, CR partners (CCR1/2, CXCR4) and corresponding heteromers. Phentolamine reduced CR:α1B/D-AR heteromers without affecting ß-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 α1-AR ligands inhibit leukocyte migration mediated by CR heteromerization partners in vivo and suggest interference with α1B-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.

Regulation of the thrombin/protease-activated receptor 1 axis by chemokine (CXC motif) receptor 4.

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 ß-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 ß-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.

Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α1-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays.

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.

KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection.

Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection.

Pharmacological modulation of C-X-C motif chemokine receptor 4 influences development of acute respiratory distress syndrome after lung ischaemia-reperfusion injury.

Activation of C-X-C motif chemokine receptor 4 (CXCR4) has been reported to result in lung protective effects in various experimental models. The effects of pharmacological CXCR4 modulation on the development of acute respiratory distress syndrome (ARDS) after lung injury, however, are unknown. Thus, we studied whether blockade and activation of CXCR4 influences development of ARDS in a unilateral lung ischaemia-reperfusion injury rat model. Anaesthetized, mechanically ventilated animals underwent right lung ischaemia (series 1, 30 minutes; series 2, 60 minutes) followed by reperfusion for 300 minutes. In series 1, animals were treated with vehicle or 0.7 µmol/kg of AMD3100 (CXCR4 antagonist) and in series 2 with vehicle, 0.7 or 3.5 µmol/kg ubiquitin (non-cognate CXCR4 agonist) within 5 minutes of reperfusion. AMD3100 significantly reduced PaO2 /FiO2 ratios, converted mild ARDS with vehicle treatment into moderate ARDS (PaO2 /FiO2 ratio<200) and increased histological lung injury. Ubiquitin dose-dependently increased PaO2 /FiO2 ratios, converted moderate-to-severe into mild-to-moderate ARDS and reduced protein content of bronchoalveolar lavage fluid (BALF). Measurements of cytokine levels (TNFα, IL-6, IL-10) in lung homogenates and BALF showed that AMD3100 reduced IL-10 levels in homogenates from post-ischaemic lungs, whereas ubiquitin dose-dependently increased IL-10 levels in BALF from post-ischaemic lungs. Our findings establish a cause-effect relationship for the effects of pharmacological CXCR4 modulation on the development of ARDS after lung ischaemia-reperfusion injury. These data further suggest CXCR4 as a new drug target to reduce the incidence and attenuate the severity of ARDS after lung injury.

Effects of the Kv7 voltage-activated potassium channel inhibitor linopirdine in rat models of haemorrhagic shock.

Recently, we demonstrated that Kv7 voltage-activated potassium channel inhibitors reduce fluid resuscitation requirements in short-term rat models of haemorrhagic shock. The aim of the present study was to further delineate the therapeutic potential and side effect profile of the Kv7 channel blocker linopirdine in various rat models of severe haemorrhagic shock over clinically relevant time periods. Intravenous administration of linopirdine, either before (1 or 3 mg/kg) or after (3 mg/kg) a 40% blood volume haemorrhage, did not affect blood pressure and survival in lethal haemorrhage models without fluid resuscitation. A single bolus of linopirdine (3 mg/kg) at the beginning of fluid resuscitation after haemorrhagic shock transiently reduced early fluid requirements in spontaneously breathing animals that were resuscitated for 3.5 hours. When mechanically ventilated rats were resuscitated after haemorrhagic shock with normal saline (NS) or with linopirdine-supplemented (10, 25 or 50 µg/mL) NS for 4.5 hours, linopirdine significantly and dose-dependently reduced fluid requirements by 14%, 45% and 55%, respectively. Lung and colon wet/dry weight ratios were reduced with linopirdine (25/50 µg/mL). There was no evidence for toxicity or adverse effects based on measurements of routine laboratory parameters and inflammation markers in plasma and tissue homogenates. Our findings support the concept that linopirdine-supplementation of resuscitation fluids is a safe and effective approach to reduce fluid requirements and tissue oedema formation during resuscitation from haemorrhagic shock.

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function.

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.

Kv7 voltage-activated potassium channel inhibitors reduce fluid resuscitation requirements after hemorrhagic shock in rats.

BACKGROUND: Recent evidence suggests that drugs targeting Kv7 channels could be used to modulate vascular function and blood pressure. Here, we studied whether Kv7 channel inhibitors can be utilized to stabilize hemodynamics and reduce resuscitation fluid requirements after hemorrhagic shock. METHODS: Anesthetized male Sprague-Dawley rats were instrumented with arterial and venous catheters for blood pressure monitoring, hemorrhage and fluid resuscitation. Series 1: Linopirdine (Kv7 channel blocker, 0.1-6 mg/kg) or retigabine (Kv7 channel activator, 0.1-12 mg/kg) were administered to normal animals. Series 2: Animals were hemorrhaged to a MAP of 25 mmHg for 30 min, followed by fluid resuscitation with normal saline (NS) to a MAP of 70 mmHg until t = 75 min. Animals were treated with single bolus injections of vehicle, linopirdine (1-6 mg/kg), XE-991 (structural analogue of linopirdine with higher potency for channel blockade, 1 mg/kg) prior to fluid resuscitation. Series 3: Animals were resuscitated with NS alone or NS supplemented with linopirdine (1.25-200 µg/mL). Data were analyzed with 2-way ANOVA/Bonferroni post-hoc testing. RESULTS: Series 1: Linopirdine transiently (10-15 min) and dose-dependently increased MAP by up to 15%. Retigabine dose-dependently reduced MAP by up to 60%, which could be reverted with linopirdine. Series 2: Fluid requirements to maintain MAP at 70 mmHg were 65 ± 34 mL/kg with vehicle, and 57 ± 13 mL/kg, 22 ± 8 mL/kg and 22 ± 11 mL/kg with intravenous bolus injection of 1, 3 and 6 mg/kg linopirdine, respectively. XE-991 (1 mg/kg), reduced resuscitation requirements comparable to 3 mg/kg linopirdine. Series 3: When resuscitation was performed with linopirdine-supplemented normal saline (NS), fluid requirements to stabilize MAP were 73 ± 12 mL/kg with NS alone and 72 ± 24, 61 ± 20, 36 ± 9 and 31 ± 9 mL/kg with NS supplemented with 1.25, 6.25, 12.5 and 200 µg/mL linopirdine, respectively. CONCLUSIONS: Our data suggest that Kv7 channel blockers could be used to stabilize blood pressure and reduce fluid resuscitation requirements after hemorrhagic shock.

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists.

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In ß-arrestin recruitment assays, ubiquitin did not sufficiently recruit ß-arrestin2 to CXCR4 (EC50 > 10 µM), whereas the EC50 for CXCL12 was 4.6 nM (95% confidence interval 3.1-6.1 nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents.

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled ß-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

New Insights into Mechanisms and Functions of Chemokine (C-X-C Motif) Receptor 4 Heteromerization in Vascular Smooth Muscle.

Recent evidence suggests that C-X-C chemokine receptor type 4 (CXCR4) heteromerizes with α1A/B-adrenoceptors (AR) and atypical chemokine receptor 3 (ACKR3) and that CXCR4:α1A/B-AR heteromers are important for α1-AR function in vascular smooth muscle cells (VSMC). Structural determinants for CXCR4 heteromerization and functional consequences of CXCR4:α1A/B-AR heteromerization in intact arteries, however, remain unknown. Utilizing proximity ligation assays (PLA) to visualize receptor interactions in VSMC, we show that peptide analogs of transmembrane-domain (TM) 2 and TM4 of CXCR4 selectively reduce PLA signals for CXCR4:α1A-AR and CXCR4:ACKR3 interactions, respectively. While both peptides inhibit CXCL12-induced chemotaxis, only the TM2 peptide inhibits phenylephrine-induced Ca(2+)-fluxes, contraction of VSMC and reduces efficacy of phenylephrine to constrict isolated arteries. In a Cre-loxP mouse model to delete CXCR4 in VSMC, we observed 60% knockdown of CXCR4. PLA signals for CXCR4:α1A/B-AR and CXCR4:ACKR3 interactions in VSMC, however, remained constant. Our observations point towards TM2/4 of CXCR4 as possible contact sites for heteromerization and suggest that TM-derived peptide analogs permit selective targeting of CXCR4 heteromers. A molecular dynamics simulation of a receptor complex in which the CXCR4 homodimer interacts with α1A-AR via TM2 and with ACKR3 via TM4 is presented. Our findings further imply that CXCR4:α1A-AR heteromers are important for intrinsic α1-AR function in intact arteries and provide initial and unexpected insights into the regulation of CXCR4 heteromerization in VSMC.

Differential protein kinase C-dependent modulation of Kv7.4 and Kv7.5 subunits of vascular Kv7 channels.

The Kv7 family (Kv7.1-7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.

Chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3 regulate vascular α1-adrenergic receptor function.

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca(2+) channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.

CXC chemokine receptor 4 signaling upon co-activation with stromal cell-derived factor-1α and ubiquitin.

Recently, we reported that extracellular ubiquitin functions as another agonist of CXC chemokine receptor (CXCR)4. Whereas the cognate CXCR4 ligand, stromal cell-derived factor (SDF)-1α, is also a CXCR7 agonist, ubiquitin does not bind to CXCR7. Because both ligands are present in the extracellular environment, co-activation of CXCR4 appears to be physiologically relevant. CXCR4 mediated effects of ubiquitin, however, are not well understood and consequences of co-activation of CXCR4 with both ligands are unknown. Utilizing proximity ligation assays and flow cytometry, we detected CXCR4, but not CXCR7, on the cell surface of THP-1 cells, which suggests that confounding effects of CXCR7 are unlikely. Time course and magnitude of reduction of cell surface CXCR4 expression were comparable after stimulation of THP-1 cells with both ligands. SDF-1α was more efficacious than ubiquitin to mobilize Ca(2+). Co-stimulation of THP-1 cells with both ligands resulted in synergistic effects on Ca(2+) fluxes at suboptimal ligand concentrations. Homologous desensitization of Ca(2+) fluxes was detectable with both ligands. SDF-1α pre-stimulation desensitized ubiquitin induced Ca(2+) fluxes, but not vice versa. Effects of SDF-1α and ubiquitin on cAMP levels, Akt and ERK1/2 phosphorylation and chemotactic responses were additive. The chemotactic activities of ubiquitin and SDF-1α were sensitive to AMD3100, pertussis toxin, U73122, LY94002 and U0126. These data suggest that CXCR4 activation with SDF-1α and ubiquitin results in partially synergistic effects on cellular signaling events and in differential effects on receptor desensitization. The ligand ratio that is present in the extracellular environment may contribute to the regulation of CXCR4 mediated functions.

Chemokine receptor hetero-oligomers regulate monocyte chemotaxis.

It is known that stress influences immune cell function. The underlying molecular mechanisms are unclear. We recently reported that many chemokine receptors (CRs) heteromerize with α1-adrenoceptors (α1-ARs) through which CRs are regulated. Here, we show that arginine vasopressin receptor 1A (AVPR1A) heteromerizes with all human CRs, except chemokine (C-X-C motif) receptor (CXCR)1, in recombinant systems and that such heteromers are detectable in THP-1 cells and human monocytes. We demonstrate that ligand-free AVPR1A differentially regulates the efficacy of CR partners to mediate chemotaxis and that AVPR1A ligands disrupt AVPR1A:CR heteromers, which enhances chemokine (C-C motif) receptor (CCR)1-mediated chemotaxis and inhibits CCR2-, CCR8-, and CXCR4-mediated chemotaxis. Using bioluminescence resonance energy transfer to monitor G protein activation and CRISPR/Cas9 gene-edited THP-1 cells lacking AVPR1A or α1B-AR, we show that CRs that share the propensity to heteromerize with α1B/D-ARs and AVPR1A exist and function within interdependent hetero-oligomeric complexes through which the efficacy of CRs to mediate chemotaxis is controlled. Our findings suggest that hetero-oligomers composed of CRs, α1B/D-ARs, and AVPR1A may enable stress hormones to regulate immune cell trafficking.

Modulation of the CXC chemokine receptor 4 agonist activity of ubiquitin through C-terminal protein modification.

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.

Regulation of the proteasome by ATP: implications for ischemic myocardial injury and donor heart preservation.

Several lines of evidence suggest that proteasomes are involved in multiple aspects of myocardial physiology and pathology, including myocardial ischemia-reperfusion injury. It is well established that the 26S proteasome is an ATP-dependent enzyme and that ischemic heart disease is associated with changes in the ATP content of the cardiomyocyte. A functional link between the 26S proteasome, myocardial ATP concentrations, and ischemic cardiac injury, however, has been suggested only recently. This review discusses the currently available data on the pathophysiological role of the cardiac proteasome during ischemia and reperfusion in the context of the cellular ATP content. Depletion of the myocardial ATP content during ischemia appears to activate the 26S proteasome via direct regulatory effects of ATP on 26S proteasome stability and activity. This implies pathological degradation of target proteins by the proteasome and could provide a pathophysiological basis for beneficial effects of proteasome inhibitors in various models of myocardial ischemia. In contrast to that in the ischemic heart, reduced and impaired proteasome activity is detectable in the postischemic heart. The paradoxical findings that proteasome inhibitors showed beneficial effects when administered during reperfusion in some studies could be explained by their anti-inflammatory and immune suppressive actions, leading to reduction of leukocyte-mediated myocardial reperfusion injury. The direct regulatory effects of ATP on the 26S proteasome have implications for the understanding of the contribution of the 26S proteasome to the pathophysiology of the ischemic heart and its possible role as a therapeutic target.

G protein activation via chemokine (C-X-C motif) receptor 4 and α1b -adrenoceptor is ligand and heteromer-dependent.

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 ß-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.

Effects of chemokine (C-C motif) receptor 2 and 3 antagonists in rat models of hemorrhagic shock.

Systemic concentrations of chemokine CCL2, an agonist at chemokine receptors CCR2/3/5, have been associated with hemodynamic instability after traumatic-hemorrhagic shock. We reported previously that the CCR2 antagonist INCB3284 prevents cardiovascular collapse and reduces fluid requirements after 30min of hemorrhagic shock (HS), whereas the CCR5 antagonist Maraviroc was ineffective. The effects of CCR3 blockade after HS are unknown and information on the therapeutic potential of INCB3284 after longer periods of HS and in HS models in the absence of fluid resuscitation (FR) is lacking. The aims of the present study were to assess the effects of CCR3 blockade with SB328437 and to further define the therapeutic efficacy of INCB3284. In series 1-3, Sprague-Dawley rats were hemorrhaged to a mean arterial blood pressure (MAP) of 30mmHg, followed by FR to MAP of 60mmHg or systolic blood pressure of 90mmHg. Series 1: 30min HS and FR until t = 90min. SB328437 at t = 30min dose-dependently reduced fluid requirements by >60%. Series 2: 60min HS and FR until t = 300min. INCB3284 and SB328437 at t = 60min reduced fluid requirements by more than 65% (p<0.05 vs. vehicle) and 25% (p>0.05 vs. vehicle), respectively, until t = 220min. Thereafter, all animals developed a steep increase in fluid requirements. Median survival time was 290min with SB328437 and >300min after vehicle and INCB3284 treatment (p<0.05). Series 3: HS/FR as in series 2. INCB3284 at t = 60min and t = 200min reduced fluid requirements by 75% until t = 300min (p<0.05 vs. vehicle). Mortality was 70% with vehicle and zero with INCB3284 treatment (p<0.05). Series 4: INCB3284 and SB328437 did not affect survival time in a lethal HS model without FR. Our findings further support the assumption that blockade of the major CCL2 receptor CCR2 is a promising approach to improve FR after HS and document that the dosing of INCB3284 can be optimized.