Limits...
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.

Show MeSH

Related in: MedlinePlus

All US28 ligands are capable of activating FAK and inducing Actin Stress Fiber Formation in reconstituted FAK−/− cells.(A) FAK activation was determined by Grb2/FAK co-immunoprecipitation reactions on Ad-FAK reconstituted FAK−/− cells infected with Ad-US28 that were treated with RANTES, Fractalkine, MCP-1. Cells were harvested in modified RIPA buffer at 0 (unstimulated), 5, 10, 15, and 30 minutes post addition of ligand. Active FAK associated with Grb2 was visualized by western blotting for phospho-FAK. (B) FAK  cells infected with Ad-US28 were reconstituted with WT FAK via adenovirus transduction. RANTES, MCP-1, or Fractalkine treated cells were fixed two hours post addition of ligand. Cells were stained for actin with phalloidin (actin) and FAK using antibodies directed against the FAK-N'terminal HA-tag, and US28 using antibodies directed against the N-terminal Flag epitope present on US28. All images are 60× magnification.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2637432&req=5

ppat-1000304-g002: All US28 ligands are capable of activating FAK and inducing Actin Stress Fiber Formation in reconstituted FAK−/− cells.(A) FAK activation was determined by Grb2/FAK co-immunoprecipitation reactions on Ad-FAK reconstituted FAK−/− cells infected with Ad-US28 that were treated with RANTES, Fractalkine, MCP-1. Cells were harvested in modified RIPA buffer at 0 (unstimulated), 5, 10, 15, and 30 minutes post addition of ligand. Active FAK associated with Grb2 was visualized by western blotting for phospho-FAK. (B) FAK cells infected with Ad-US28 were reconstituted with WT FAK via adenovirus transduction. RANTES, MCP-1, or Fractalkine treated cells were fixed two hours post addition of ligand. Cells were stained for actin with phalloidin (actin) and FAK using antibodies directed against the FAK-N'terminal HA-tag, and US28 using antibodies directed against the N-terminal Flag epitope present on US28. All images are 60× magnification.

Mentions: To determine if the different phenotypic outcomes of RANTES or Fractalkine binding to US28 is reflected in differences at the level of signal transduction, we examined the ability each class of chemokine ligand to activate FAK through binding to US28. We have previously demonstrated that RANTES binding to US28 stimulates the activation of FAK, promoting a specific association between phosphorylated FAK and the adaptor protein Grb2. FAK is a critical mediator of focal adhesion turnover and plays important roles in cellular adhesion and motility. As such, it displays high basal activity levels in most cell types. For these experiments we developed a clean inducible signaling assay using FAK knockout mouse fibroblasts (FAK−/−) that have been reconstituted with an adenovirus vector expressing wild-type FAK concurrent with the addition of Ad-US28 [16]. To determine the ability of CC-chemokines and the CX3C-chemokine Fractalkine to promote US28 mediated activation of FAK and formation of active Grb2/FAK complexes, FAK−/− cells expressing US28 alone or in combination with FAK were stimulated with RANTES, MCP-1 or Fractalkine (40ng/ml) for 0 (unstimulated), 5, 10, 15 or 30 minutes. Grb2 was immunoprecipitated and active FAK associated with Grb2 visualized by western blotting for Phospho-Tyr [16]. RANTES, MCP-1 and Fractalkine all promoted US28-mediated FAK activation and formation of Grb2/FAK complexes with similar kinetics but slightly different magnitudes of activation (Figure 2A).


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)

All US28 ligands are capable of activating FAK and inducing Actin Stress Fiber Formation in reconstituted FAK−/− cells.(A) FAK activation was determined by Grb2/FAK co-immunoprecipitation reactions on Ad-FAK reconstituted FAK−/− cells infected with Ad-US28 that were treated with RANTES, Fractalkine, MCP-1. Cells were harvested in modified RIPA buffer at 0 (unstimulated), 5, 10, 15, and 30 minutes post addition of ligand. Active FAK associated with Grb2 was visualized by western blotting for phospho-FAK. (B) FAK  cells infected with Ad-US28 were reconstituted with WT FAK via adenovirus transduction. RANTES, MCP-1, or Fractalkine treated cells were fixed two hours post addition of ligand. Cells were stained for actin with phalloidin (actin) and FAK using antibodies directed against the FAK-N'terminal HA-tag, and US28 using antibodies directed against the N-terminal Flag epitope present on US28. All images are 60× magnification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637432&req=5

ppat-1000304-g002: All US28 ligands are capable of activating FAK and inducing Actin Stress Fiber Formation in reconstituted FAK−/− cells.(A) FAK activation was determined by Grb2/FAK co-immunoprecipitation reactions on Ad-FAK reconstituted FAK−/− cells infected with Ad-US28 that were treated with RANTES, Fractalkine, MCP-1. Cells were harvested in modified RIPA buffer at 0 (unstimulated), 5, 10, 15, and 30 minutes post addition of ligand. Active FAK associated with Grb2 was visualized by western blotting for phospho-FAK. (B) FAK cells infected with Ad-US28 were reconstituted with WT FAK via adenovirus transduction. RANTES, MCP-1, or Fractalkine treated cells were fixed two hours post addition of ligand. Cells were stained for actin with phalloidin (actin) and FAK using antibodies directed against the FAK-N'terminal HA-tag, and US28 using antibodies directed against the N-terminal Flag epitope present on US28. All images are 60× magnification.
Mentions: To determine if the different phenotypic outcomes of RANTES or Fractalkine binding to US28 is reflected in differences at the level of signal transduction, we examined the ability each class of chemokine ligand to activate FAK through binding to US28. We have previously demonstrated that RANTES binding to US28 stimulates the activation of FAK, promoting a specific association between phosphorylated FAK and the adaptor protein Grb2. FAK is a critical mediator of focal adhesion turnover and plays important roles in cellular adhesion and motility. As such, it displays high basal activity levels in most cell types. For these experiments we developed a clean inducible signaling assay using FAK knockout mouse fibroblasts (FAK−/−) that have been reconstituted with an adenovirus vector expressing wild-type FAK concurrent with the addition of Ad-US28 [16]. To determine the ability of CC-chemokines and the CX3C-chemokine Fractalkine to promote US28 mediated activation of FAK and formation of active Grb2/FAK complexes, FAK−/− cells expressing US28 alone or in combination with FAK were stimulated with RANTES, MCP-1 or Fractalkine (40ng/ml) for 0 (unstimulated), 5, 10, 15 or 30 minutes. Grb2 was immunoprecipitated and active FAK associated with Grb2 visualized by western blotting for Phospho-Tyr [16]. RANTES, MCP-1 and Fractalkine all promoted US28-mediated FAK activation and formation of Grb2/FAK complexes with similar kinetics but slightly different magnitudes of activation (Figure 2A).

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