Muscarinic receptor reserve for inhibition of cAMP accumulation in bovine trachealis cells

Ethier, Michael F.; Schaefer, Oren P.; Samant, Neha S.; Yamaguchi, Hiroshi; Madison, J. Mark

doi:10.1152/ajplung.1996.270.2.l199

Cited by 7 publications

(5 citation statements)

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“…This suggests that occupancy of only a small fraction of receptors by ubiquitin evokes a cellular response. Such a doseresponse relationship has been described for other G proteincoupled receptors, which showed half-maximal and maximal responses at receptor occupancies of 0.13 and 0.8%, respectively (47,48). In addition, our finding corresponds to the previous observation that intramuscular injection of ubiquitin led…”

Section: Discussionsupporting

confidence: 90%

The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions

Saini

Staren

Ziarek

et al. 2011

Journal of Biological Chemistry

123

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Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1␣ do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1␣ lead to similar G␣ iresponses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-l␣ in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1␣, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.

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Section: Discussionsupporting

confidence: 90%

The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions

Saini

Staren

Ziarek

et al. 2011

Journal of Biological Chemistry

123

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“…Despite activities of the disulfide-locked dimeric CXCL12 variant CXCL12 2 in β-arrestin 2 recruitment assays for CXCR4 and ACKR3 that were comparable with CXCL12α/β, CXCL12 2 showed significantly reduced potency to attenuate thrombin-induced permeability of hPPEAC. The previous finding that CXCL12 2 binds to ACKR3 with very low affinity is not contradictive to our findings in ACKR3 β-arrestin 2 recruitment assays because maximal biological responses of other GPCRs have been observed at ligand occupancies of only a small fraction of receptors [ 28 , 52 , 53 ] and a large receptor reserve is likely in expression systems, such as the Presto-Tango assay. The effects of CXC12 2 in CXCR4 β-arrestin 2 recruitment assays that we observed using the Presto-Tango cell system, however, are conflicting with previous measurements in intermolecular bioluminescence resonance energy transfer (BRET) assays [ 28 ].…”

Section: Discussioncontrasting

confidence: 55%

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

et al. 2017

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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.

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“…When tested in pressure myography experiments, CXCL11 and CXCL12 attenuated PE- and aVP-induced vasoconstriction to a similar degree ( p < 0.05 for vehicle versus CXCL11 and CXCL12; p > 0.05 for CXCL11 versus CXCL12), whereas CXCL11 (3–73) did not ( figure 1 d , e ). The previous findings that CXCL12 binds to ACKR3 with sevenfold to 20-fold higher affinity than CXCL11 are not contradictive to our observations, because maximal biological responses of other GPCRs have been observed at ligand occupancies of only a small fraction of receptors [ 29 – 32 ]. In addition, our findings are consistent with the recent observation that the potency of CXCL12 to induce β-arrestin recruitment to ACKR3 when measured in a bioluminescence resonance energy transfer (BRET)-based assay was twofold higher than that of CXCL11 [ 30 ].…”

Section: Resultscontrasting

confidence: 46%

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle

et al. 2018

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Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

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Muscarinic receptor reserve for inhibition of cAMP accumulation in bovine trachealis cells

Cited by 7 publications

References 0 publications

The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions

The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle

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