Selective Elimination of High Constitutive Activity or Chemokine Binding in the Human Herpesvirus 8 Encoded Seven Transmembrane Oncogene ORF74

Mette M Rosenkilde,Thomas N Kledal,Peter J Holst,Thue W Schwartz

doi:10.1074/jbc.m003800200

Mette M Rosenkilde, Thomas N Kledal + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m003800200

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Abstract

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

Highlights

Chemokines are chemotactic cytokines that regulate immunological processes through interaction with 7TM1 G-proteincoupled receptors expressed mainly on leukocytes
Through targeted mutagenesis we here create three distinct phenotypes of Open reading frame 74 (ORF74): a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II
It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity

Summary

The abbreviations used are

Through an ancient act of molecular piracy and are structurally optimized for a particular pharmacological phenotype of benefit to the virus. ORF74 from HHV-8 binds various human CXC chemokines [19, 20] The properties of these chemokines on ORF74 signaling cover the whole pharmacological spectrum: GRO␣, GRO␤ and GRO␥ are agonists, IP-10, stromal cell-derived factor-1␣, granulocyte colony stimulating factor-2, and vMIP-II are inverse agonists, whereas the inflammatory CXC chemokines IL-8, neutrophil-activating peptide-2, and epithelial cell-derived activating peptide-78 are neutral ligands, which despite high affinity binding do not affect signaling of the receptor [20]. The present study is aimed at trying to characterize, in the ORF74 receptor from HHV-8, the structural basis for its broad spectrum binding profile of chemokines as well as the structural basis for its high constitutive activity based on knowledge on ligand binding and signaling properties in other 7TM receptors. Thereby, we created specific ORF74 mutants in which certain elements of the pharmacological repertoire have been selectively eliminated; to exploit these mutants in future transgenic studies to identify the molecular mechanism that HHV-8 has exploited in ORF74 to precipitate the different clinical features of Kaposi’s sarcoma and other HHV-8-related malignancies

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