ABSTRACT
The human cytomegalovirus-encoded G protein-coupled receptor homologue US28 binds inflammatory chemokines and sequesters them from the environment of infected cells. Low surface deposition and endocytosis are dependent on constitutive C-terminal phosphorylation, suggesting a requirement for beta-arrestin binding in receptor internalization. In this report, a US28-dependent redistribution of beta-arrestin into vesicular structures occurred, although internalization of US28 was independent of beta-arrestin. Internalization of US28 was dynamin-dependent, and US28 partially partitioned into the detergent-resistant membrane fraction. Endocytosis was diminished by cholesterol depletion, yet sucrose inhibition was even stronger. The relevance of the clathrin-coated pit pathway was supported by colocalization of beta(2)-adaptin and US28 in endocytic compartments. Exchange of the C-terminal dileucine endocytosis motif inhibited rapid endocytosis, indicating a direct interaction of US28 with the AP-2 adaptor complex. We suggest that the arrestin-independent, dynamin-dependent internalization of US28 reveals a differential sorting of beta-arrestins and the virally encoded chemokine receptor homologue.
Subject(s)
Arrestins/metabolism , Caveolae/metabolism , Dynamins/metabolism , Endocytosis/physiology , Kidney/cytology , Kidney/metabolism , Receptors, Chemokine/metabolism , Viral Proteins/metabolism , Caveolae/ultrastructure , Cell Line , Humans , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Mutagenesis, Site-Directed , Recombinant Proteins/metabolism , Signal Transduction/physiology , Tissue Distribution , beta-ArrestinsABSTRACT
Human cytomegalovirus encodes the G protein-coupled chemokine receptor homologue US28 that binds several CC chemokines and sequesters extracellular chemokines from the environment of infected cells. Mechanistically, it has been shown that US28 undergoes rapid constitutive receptor endocytosis and recycling. Monoclonal antibodies were raised that allowed the characterization of a ligand-independent phosphorylation and low surface expression of the US28 receptor in transiently transfected HEK293A cells. Phosphoamino acid analysis defined C-terminal serine and threonine residues as phospho-acceptor sites for constitutive receptor phosphorylation. Coexpression of G protein-coupled receptor kinase-2 and US28 enhanced ligand-independent receptor phosphorylation. C-terminal serine to alanine mutagenesis of US28 resulted in a decreased phosphorylation rate that correlated with enhanced surface expression. Maximal surface expression was detected when all C-terminal serines were substituted. Exchange of all C-terminal serines also significantly reduced receptor endocytosis. Thus, constitutive US28 phosphorylation regulates receptor endocytosis and receptor surface display and may thereby provide a pathogenic mechanism for a potential decoy function of the virally encoded receptor.
Subject(s)
Cell Membrane/metabolism , Cytomegalovirus/metabolism , Endocytosis/physiology , Receptors, Chemokine/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Cattle , Cell Line , Chemokine CCL5/metabolism , Enzyme Inhibitors/metabolism , Fibroblasts/cytology , Fibroblasts/physiology , Fibroblasts/virology , Genes, Reporter , Humans , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-kappa B/metabolism , Phosphorylation , Receptors, Chemokine/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Viral Proteins/geneticsABSTRACT
CD30 is selectively expressed on the tumor cells of a variety of malignant disorders of the immune system and can therefore be used as a target for an anti-CD30 antibody-based immunotherapy. However, CD30 is cleaved at the cell surface by tumor necrosis factor-alpha converting enzyme (TACE). This metalloproteinase releases the soluble ectodomain of CD30 (sCD30), which is able to neutralize immunotherapeutic agents before these reach their target cells. Such constitutive CD30 cleavage is enhanced after binding of most anti-CD30 antibodies, leading to a downregulation of CD30 and an increase of sCD30 in the cell environment. Here, we demonstrate that CD30 shedding from the cell line Karpas 299 could effectively be blocked by the hydroxamic acid-based metalloproteinase inhibitors BB-3644 (IC50 = 180 nM), BB-2116 (IC50 = 230 nM), BB-94 (batimastat, IC50 = 230 nM) and BB-2516 (marimastat, IC50 = 1 microM). This inhibition reduced the concentration of sCD30 in the cell environment to the background level, prolonged the persistence of the anti-CD30 antibody Ki-3 on Karpas 299 cells and favored its internalization. Moreover, a nontoxic concentration of the inhibitor BB-3644 significantly increased the cytotoxic activity of the anti-CD30 ricin A-chain immunotoxin Ki-3.dgA towards the CD30(+) Hodgkin-derived cell line L540. Hence, the metalloproteinase inhibitor BB-3644 may be a promising compound to improve the immunotherapy of CD30(+) malignancies.
