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Differential ligand binding to a human cytomegalovirus chemokine receptor determines cell type-specific motility.

Vomaske J, Melnychuk RM, Smith PP, Powell J, Hall L, DeFilippis V, Früh K, Smit M, Schlaepfer DD, Nelson JA, Streblow DN - PLoS Pathog. (2009)

Bottom Line: Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages.These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types.Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type-specific has important implications in the role of US28 in HCMV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology and The Vaccine and Gene Therapy Institute, Oregon Health & Science University, Portland, OR, USA.

ABSTRACT
While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX(3)C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Galpha12 and Fractalkine through Galphaq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type-specific has important implications in the role of US28 in HCMV pathogenesis.

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Fractalkine inhibits US28-mediated SMC migration induced by RANTES.(A) SMC migration assays were performed on cells infected with adenovirus expressing US28-HA treated with either RANTES or Fractalkine at the indicated concentrations. Data are represented as a percentage of unstimulated cells infected with control adenovirus transactivator only. For all conditions, n>6 from two independent experiments. (B) SMC migration assays were performed on US28-expressing cells treated with RANTES, Fractalkine or 40ng/ml of RANTES and the indicated concentrations of Fractalkine as a competing ligand.
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ppat-1000304-g001: Fractalkine inhibits US28-mediated SMC migration induced by RANTES.(A) SMC migration assays were performed on cells infected with adenovirus expressing US28-HA treated with either RANTES or Fractalkine at the indicated concentrations. Data are represented as a percentage of unstimulated cells infected with control adenovirus transactivator only. For all conditions, n>6 from two independent experiments. (B) SMC migration assays were performed on US28-expressing cells treated with RANTES, Fractalkine or 40ng/ml of RANTES and the indicated concentrations of Fractalkine as a competing ligand.

Mentions: The unique ability of US28 to bind both CC- and CX3C-chemokine ligands raises the question of whether US28 signaling is not only ligand-dependent, but also ligand-specific [9],[13],[20],[21]. To determine whether US28 signaling and SMC migration are ligand-specific, we performed SMC migration and signaling assays on US28 adenovirus expressing primary rat SMC in the presence of RANTES or Fractalkine. In this assay, RANTES readily induced US28-mediated SMC migration, however, increasing concentrations of Fractalkine failed to stimulate cellular motility above Ad-tet-transactivator (Trans) infected and RANTES stimulated controls, indicating that not all US28 ligands evoke the same functional response (Figure 1A). Visual analysis of the cells prior to and following the migration assay indicated that the lack of migration was not due to overt cell death mediated by US28 expression and subsequent treatment with Fractalkine (data not shown). A competition assay was performed to determine whether Fractalkine inhibits the ability of RANTES to induce SMC migration. In these experiments, RANTES alone promoted SMC migration, as expected. However, Fractalkine, at concentrations as low as 10ng/ml, was sufficient to block RANTES-mediated SMC migration (Figure 1B) suggesting that Fractalkine is a competitive inhibitor to CC-chemokine induced SMC migration.


Differential ligand binding to a human cytomegalovirus chemokine receptor determines cell type-specific motility.

Vomaske J, Melnychuk RM, Smith PP, Powell J, Hall L, DeFilippis V, Früh K, Smit M, Schlaepfer DD, Nelson JA, Streblow DN - PLoS Pathog. (2009)

Fractalkine inhibits US28-mediated SMC migration induced by RANTES.(A) SMC migration assays were performed on cells infected with adenovirus expressing US28-HA treated with either RANTES or Fractalkine at the indicated concentrations. Data are represented as a percentage of unstimulated cells infected with control adenovirus transactivator only. For all conditions, n>6 from two independent experiments. (B) SMC migration assays were performed on US28-expressing cells treated with RANTES, Fractalkine or 40ng/ml of RANTES and the indicated concentrations of Fractalkine as a competing ligand.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637432&req=5

ppat-1000304-g001: Fractalkine inhibits US28-mediated SMC migration induced by RANTES.(A) SMC migration assays were performed on cells infected with adenovirus expressing US28-HA treated with either RANTES or Fractalkine at the indicated concentrations. Data are represented as a percentage of unstimulated cells infected with control adenovirus transactivator only. For all conditions, n>6 from two independent experiments. (B) SMC migration assays were performed on US28-expressing cells treated with RANTES, Fractalkine or 40ng/ml of RANTES and the indicated concentrations of Fractalkine as a competing ligand.
Mentions: The unique ability of US28 to bind both CC- and CX3C-chemokine ligands raises the question of whether US28 signaling is not only ligand-dependent, but also ligand-specific [9],[13],[20],[21]. To determine whether US28 signaling and SMC migration are ligand-specific, we performed SMC migration and signaling assays on US28 adenovirus expressing primary rat SMC in the presence of RANTES or Fractalkine. In this assay, RANTES readily induced US28-mediated SMC migration, however, increasing concentrations of Fractalkine failed to stimulate cellular motility above Ad-tet-transactivator (Trans) infected and RANTES stimulated controls, indicating that not all US28 ligands evoke the same functional response (Figure 1A). Visual analysis of the cells prior to and following the migration assay indicated that the lack of migration was not due to overt cell death mediated by US28 expression and subsequent treatment with Fractalkine (data not shown). A competition assay was performed to determine whether Fractalkine inhibits the ability of RANTES to induce SMC migration. In these experiments, RANTES alone promoted SMC migration, as expected. However, Fractalkine, at concentrations as low as 10ng/ml, was sufficient to block RANTES-mediated SMC migration (Figure 1B) suggesting that Fractalkine is a competitive inhibitor to CC-chemokine induced SMC migration.

Bottom Line: Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages.These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types.Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type-specific has important implications in the role of US28 in HCMV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology and The Vaccine and Gene Therapy Institute, Oregon Health & Science University, Portland, OR, USA.

ABSTRACT
While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX(3)C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Galpha12 and Fractalkine through Galphaq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type-specific has important implications in the role of US28 in HCMV pathogenesis.

Show MeSH
Related in: MedlinePlus