Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74.
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.
Rosenkilde
M M
MM
Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Kledal
T N
TN
Holst
P J
PJ
Schwartz
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D018925:Chemokines / Q000378:metabolism*; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018925:Chemokines; C105181:ORF74 protein, Human herpesvirus 8; D019707:Receptors, Chemokine; D014764:Viral Proteins