TY - JOUR

T1 - The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis

AU - Waldhoer, Maria

AU - Casarosa, Paola

AU - Rosenkilde, Mette M

AU - Smit, Martine J

AU - Leurs, Rob

AU - Whistler, Jennifer L

AU - Schwartz, Thue W

N1 - Keywords: Amino Acid Sequence; Animals; COS Cells; Chemokine CX3CL1; Chemokines, CX3C; Cyclic AMP Response Element-Binding Protein; Cytoplasm; Endocytosis; Gene Deletion; Gene Expression; Inositol Phosphates; Iodine Radioisotopes; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; NF-kappa B; Radioligand Assay; Receptors, Chemokine; Receptors, Neurokinin-1; Recombinant Fusion Proteins; Signal Transduction; Structure-Activity Relationship; Transcription, Genetic; Transfection; Viral Proteins

PY - 2003

Y1 - 2003

N2 - US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show that the cytoplasmic tail domain of US28 per se regulates receptor endocytosis, independent of the signaling ability of the core domain of US28. The constitutive endocytic property of the US28 c-tail was transposable to other 7TM receptors, the herpes virus 8-encoded ORF74 and the tachykinin NK1 receptor (ORF74-US28-ctail and NK1-US28-ctail). Deletion of the US28 C terminus resulted in reduced constitutive endocytosis and consequently enhanced signaling capacity of all receptors tested as assessed by inositol phosphate turnover, NF-kappa B, and cAMP-responsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM receptor can camouflage the agonist properties of a ligand.

AB - US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show that the cytoplasmic tail domain of US28 per se regulates receptor endocytosis, independent of the signaling ability of the core domain of US28. The constitutive endocytic property of the US28 c-tail was transposable to other 7TM receptors, the herpes virus 8-encoded ORF74 and the tachykinin NK1 receptor (ORF74-US28-ctail and NK1-US28-ctail). Deletion of the US28 C terminus resulted in reduced constitutive endocytosis and consequently enhanced signaling capacity of all receptors tested as assessed by inositol phosphate turnover, NF-kappa B, and cAMP-responsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM receptor can camouflage the agonist properties of a ligand.

U2 - 10.1074/jbc.M213179200

DO - 10.1074/jbc.M213179200

M3 - Journal article

C2 - 12646575

VL - 278

SP - 19473

EP - 19482

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -