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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

Characterization and β-arrestin recruitment to ORF74-R3.50A.(A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R3.50A (R3.50A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R3.50A-Rluc8 (R3.50A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R3.50A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).
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pone.0124486.g003: Characterization and β-arrestin recruitment to ORF74-R3.50A.(A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R3.50A (R3.50A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R3.50A-Rluc8 (R3.50A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R3.50A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).

Mentions: The conserved DRY motif (Asp-Arg-Tyr), located at the boundary between transmembrane domain 3 and intracellular loop 2 of GPCRs, is required for G protein activation. Mutation of the highly conserved R3.50 (Ballesteros-Weinstein numbering [17]) impairs G protein-dependent signaling of many GPCRs [30], including ORF74 [31–33]. ORF74-R3.50A was expressed at the cell surface at levels 1.3-fold higher compared to WT-ORF74 as determined by ELISA (Fig 3A), but completely lost its ability to constitutively activate PLC (Fig 3B) and showed no CXCL1- or CXCL8-agonism (S2 Fig) as previously reported [32]. Although unable to activate PLC signaling, ORF74-R3.50A recruited both β-arrestin1 (Fig 3C) and β-arrestin2 (Fig 3D) in response to CXCL1 with potencies (pEC50 values are 8.5 ± 0.1 and 8.8 ± 0.1, respectively) that did not significantly differ from WT-ORF74 (see Table 1).


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)

Characterization and β-arrestin recruitment to ORF74-R3.50A.(A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R3.50A (R3.50A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R3.50A-Rluc8 (R3.50A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R3.50A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).
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pone.0124486.g003: Characterization and β-arrestin recruitment to ORF74-R3.50A.(A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R3.50A (R3.50A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R3.50A-Rluc8 (R3.50A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R3.50A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).
Mentions: The conserved DRY motif (Asp-Arg-Tyr), located at the boundary between transmembrane domain 3 and intracellular loop 2 of GPCRs, is required for G protein activation. Mutation of the highly conserved R3.50 (Ballesteros-Weinstein numbering [17]) impairs G protein-dependent signaling of many GPCRs [30], including ORF74 [31–33]. ORF74-R3.50A was expressed at the cell surface at levels 1.3-fold higher compared to WT-ORF74 as determined by ELISA (Fig 3A), but completely lost its ability to constitutively activate PLC (Fig 3B) and showed no CXCL1- or CXCL8-agonism (S2 Fig) as previously reported [32]. Although unable to activate PLC signaling, ORF74-R3.50A recruited both β-arrestin1 (Fig 3C) and β-arrestin2 (Fig 3D) in response to CXCL1 with potencies (pEC50 values are 8.5 ± 0.1 and 8.8 ± 0.1, respectively) that did not significantly differ from WT-ORF74 (see Table 1).

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