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The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

de Munnik SM, Kooistra AJ, van Offenbeek J, Nijmeijer S, de Graaf C, Smit MJ, Leurs R, Vischer HF - PLoS ONE (2015)

Bottom Line: Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74.Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking.Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Pharmaceutical Sciences, Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), VU University Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

No MeSH data available.


Related in: MedlinePlus

Serines and threonines at the distal end of the C-tail are essential for β-arrestin recruitment.(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine are shown in bold brown and clustered to indicate the different ORF74-ST/A mutants (ST/A1, ST/A2 and ST/A3). The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [35]. (B) HEK293T cells were transiently transfected with ORF74-Rluc8 (WT), ORF74-ST/A1-Rluc8 (ST/A1), ORF74-ST/A2-Rluc8 (ST/A2) or ORF74-ST/A3-Rluc8 (ST/A3) or empty vector DNA (mock-transfected) and receptor cell surface expression was determined by ELISA. (C-F) HEK293T cells expressing ORF74-Rluc8 (WT) or one of the Rluc8-tagged ORF74-ST/A mutants in combination with β-arrestin1-eYFP (C, E) or β-arrestin2-eYFP (D, F) were treated with increasing concentrations CXCL1 (C, D) or were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) (E, F) before measurement of BRET. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over mock-transfected cells (dotted line) (B) or fold over basal (C-F) and error bars indicate SEM values. Statistical differences between ORF74 WT and mutant cell surface expression (B) or difference between vehicle- and corresponding CXCL1-treated cells (E, F) were determined by one-way ANOVA followed by a Bonferroni test (B) or a Student t test (E, F), respectively (**** p ≤ 0.0001, ** p ≤ 0.01). NS = not significant.
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pone.0124486.g005: Serines and threonines at the distal end of the C-tail are essential for β-arrestin recruitment.(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine are shown in bold brown and clustered to indicate the different ORF74-ST/A mutants (ST/A1, ST/A2 and ST/A3). The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [35]. (B) HEK293T cells were transiently transfected with ORF74-Rluc8 (WT), ORF74-ST/A1-Rluc8 (ST/A1), ORF74-ST/A2-Rluc8 (ST/A2) or ORF74-ST/A3-Rluc8 (ST/A3) or empty vector DNA (mock-transfected) and receptor cell surface expression was determined by ELISA. (C-F) HEK293T cells expressing ORF74-Rluc8 (WT) or one of the Rluc8-tagged ORF74-ST/A mutants in combination with β-arrestin1-eYFP (C, E) or β-arrestin2-eYFP (D, F) were treated with increasing concentrations CXCL1 (C, D) or were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) (E, F) before measurement of BRET. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over mock-transfected cells (dotted line) (B) or fold over basal (C-F) and error bars indicate SEM values. Statistical differences between ORF74 WT and mutant cell surface expression (B) or difference between vehicle- and corresponding CXCL1-treated cells (E, F) were determined by one-way ANOVA followed by a Bonferroni test (B) or a Student t test (E, F), respectively (**** p ≤ 0.0001, ** p ≤ 0.01). NS = not significant.

Mentions: To identify which S/T residues are involved in β-arrestin recruitment, Ala-substitutions were introduced in three different segments of the C-tail of ORF74 (ST/A1, ST/A2 and ST/A3) (Fig 5A). The Rluc8-tagged ORF74 mutants were expressed at the cell surface at levels similar to WT-ORF74-Rluc8, as determined by ELISA (Fig 5B). ORF74-ST/A1 recruited β-arrestin1 (Fig 5C) and β-arrestin2 (Fig 5D) in response to CXCL1 with potencies (pEC50 = 8.0 ± 0.1 and 8.3 ± 0.1, respectively) that did not significantly differ from WT-ORF74 (see Table 1). On the contrary, ORF74-ST/A2 completely lost its ability to recruit β-arrestin1 (Fig 5E) and β-arrestin2 (Fig 5F) in response to CXCL1. ORF74-ST/A3 was unable to recruit β-arrestin1 (Fig 5E), but showed a small increase in CXCL1-induced β-arrestin2 recruitment (Fig 5F). These results show that the two distal S/T clusters, but not the two proximal S residues in the C-terminal region of ORF74, are essential for β-arrestin1/2 recruitment.


The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

de Munnik SM, Kooistra AJ, van Offenbeek J, Nijmeijer S, de Graaf C, Smit MJ, Leurs R, Vischer HF - PLoS ONE (2015)

Serines and threonines at the distal end of the C-tail are essential for β-arrestin recruitment.(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine are shown in bold brown and clustered to indicate the different ORF74-ST/A mutants (ST/A1, ST/A2 and ST/A3). The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [35]. (B) HEK293T cells were transiently transfected with ORF74-Rluc8 (WT), ORF74-ST/A1-Rluc8 (ST/A1), ORF74-ST/A2-Rluc8 (ST/A2) or ORF74-ST/A3-Rluc8 (ST/A3) or empty vector DNA (mock-transfected) and receptor cell surface expression was determined by ELISA. (C-F) HEK293T cells expressing ORF74-Rluc8 (WT) or one of the Rluc8-tagged ORF74-ST/A mutants in combination with β-arrestin1-eYFP (C, E) or β-arrestin2-eYFP (D, F) were treated with increasing concentrations CXCL1 (C, D) or were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) (E, F) before measurement of BRET. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over mock-transfected cells (dotted line) (B) or fold over basal (C-F) and error bars indicate SEM values. Statistical differences between ORF74 WT and mutant cell surface expression (B) or difference between vehicle- and corresponding CXCL1-treated cells (E, F) were determined by one-way ANOVA followed by a Bonferroni test (B) or a Student t test (E, F), respectively (**** p ≤ 0.0001, ** p ≤ 0.01). NS = not significant.
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Related In: Results  -  Collection

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Show All Figures
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pone.0124486.g005: Serines and threonines at the distal end of the C-tail are essential for β-arrestin recruitment.(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine are shown in bold brown and clustered to indicate the different ORF74-ST/A mutants (ST/A1, ST/A2 and ST/A3). The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [35]. (B) HEK293T cells were transiently transfected with ORF74-Rluc8 (WT), ORF74-ST/A1-Rluc8 (ST/A1), ORF74-ST/A2-Rluc8 (ST/A2) or ORF74-ST/A3-Rluc8 (ST/A3) or empty vector DNA (mock-transfected) and receptor cell surface expression was determined by ELISA. (C-F) HEK293T cells expressing ORF74-Rluc8 (WT) or one of the Rluc8-tagged ORF74-ST/A mutants in combination with β-arrestin1-eYFP (C, E) or β-arrestin2-eYFP (D, F) were treated with increasing concentrations CXCL1 (C, D) or were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) (E, F) before measurement of BRET. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over mock-transfected cells (dotted line) (B) or fold over basal (C-F) and error bars indicate SEM values. Statistical differences between ORF74 WT and mutant cell surface expression (B) or difference between vehicle- and corresponding CXCL1-treated cells (E, F) were determined by one-way ANOVA followed by a Bonferroni test (B) or a Student t test (E, F), respectively (**** p ≤ 0.0001, ** p ≤ 0.01). NS = not significant.
Mentions: To identify which S/T residues are involved in β-arrestin recruitment, Ala-substitutions were introduced in three different segments of the C-tail of ORF74 (ST/A1, ST/A2 and ST/A3) (Fig 5A). The Rluc8-tagged ORF74 mutants were expressed at the cell surface at levels similar to WT-ORF74-Rluc8, as determined by ELISA (Fig 5B). ORF74-ST/A1 recruited β-arrestin1 (Fig 5C) and β-arrestin2 (Fig 5D) in response to CXCL1 with potencies (pEC50 = 8.0 ± 0.1 and 8.3 ± 0.1, respectively) that did not significantly differ from WT-ORF74 (see Table 1). On the contrary, ORF74-ST/A2 completely lost its ability to recruit β-arrestin1 (Fig 5E) and β-arrestin2 (Fig 5F) in response to CXCL1. ORF74-ST/A3 was unable to recruit β-arrestin1 (Fig 5E), but showed a small increase in CXCL1-induced β-arrestin2 recruitment (Fig 5F). These results show that the two distal S/T clusters, but not the two proximal S residues in the C-terminal region of ORF74, are essential for β-arrestin1/2 recruitment.

Bottom Line: Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74.Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking.Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Pharmaceutical Sciences, Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), VU University Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

No MeSH data available.


Related in: MedlinePlus