title: The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.
authors.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.
authors.forename: Sabrina M
authors.initials: SM
authors.lastname: de Munnik
authors.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.
authors.forename: Rosan
authors.initials: R
authors.lastname: van der Lee
authors.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.
authors.forename: Daniëlle M
authors.initials: DM
authors.lastname: Velders
authors.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.
authors.forename: Jody
authors.initials: J
authors.lastname: van Offenbeek
authors.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.
authors.forename: Laura
authors.initials: L
authors.lastname: Smits-de Vries
authors.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.
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.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.
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Smit
authors.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. Electronic address: h.f.vischer@vu.nl.
authors.forename: Henry F
authors.initials: HF
authors.lastname: Vischer
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000378:metabolism*; D019707:Receptors, Chemokine / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C105181:ORF74 protein, Human herpesvirus 8; D019707:Receptors, Chemokine; D014764:Viral Proteins; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017526:Receptor, IGF Type 1; D016392:Proto-Oncogene Proteins pp60(c-src); D010738:Type C Phospholipases
keywords: Crosstalk; G protein-coupled receptor; Herpesvirus; Insulin-like growth factor-1 receptor; ORF74; Receptor tyrosine kinase