Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

Holst, Peter Johannes; Manfra, Denise; Chen, Shu‐Cheng; Wiekowski, Maria; Holst, Birgitte; Cifire, F; Lipp, Martin; Schwartz, Thue W.; Lira, Sérgio A.

doi:10.1172/jci13622

Cited by 100 publications

(78 citation statements)

References 32 publications

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“…For phenotypical analysis, 10 6 FDGloaded cells were stained with the directly conjugated mAbs: CD31 (MEC13.3), CD34 (RAM34), CD45 (30-F11), CD11b (M1/70), and IAb (25-9-17) (BD Pharmingen). Detection of ORF74 expression in thymocytes was done according to a previously described method (27). Events (2-3 ϫ 10 5 ) were acquired on a FACScan (BD Immunocytometry Systems), and sorting was done on a Mo-Flo sorter (DakoCytomation) according to standard protocols.…”

Section: Flow Cytometrymentioning

confidence: 99%

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The Human Herpes Virus 8-Encoded Chemokine Receptor Is Required for Angioproliferation in a Murine Model of Kaposi’s Sarcoma

Jensen

Manfra²,

Grisotto³

et al. 2005

The Journal of Immunology

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Kaposi’s sarcoma (KS)-associated herpesvirus or human herpes virus 8 is considered the etiological agent of KS, a highly vascularized neoplasm that is the most common tumor affecting HIV/AIDS patients. The KS-associated herpesvirus/human herpes virus 8 open reading frame 74 encodes a constitutively active G protein-coupled receptor known as vGPCR that binds CXC chemokines with high affinity. In this study, we show that conditional transgenic expression of vGPCR by cells of endothelial origin triggers an angiogenic program in vivo, leading to development of an angioproliferative disease that resembles KS. This angiogenic program consists partly in the expression of the angiogenic factors placental growth factor, platelet-derived growth factor B, and inducible NO synthase by the vGPCR-expressing cells. Finally, we show that continued vGPCR expression is essential for progression of the KS-like phenotype and that down-regulation of vGPCR expression results in reduced expression of angiogenic factors and regression of the lesions. Together, these findings implicate vGPCR as a key element in KS pathogenesis and suggest that strategies to block its function may represent a novel approach for the treatment of KS.

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Section: Flow Cytometrymentioning

confidence: 99%

“…1A). We used the human CD2 promoter to control rtTA expression, because we have previously reported that transgenic mice expressing vGPCR under control of this promoter develop angioproliferative lesions in vivo (21,27).…”

Section: Transgenic Mice Expressing Vgpcr Conditionally Develop Kslikmentioning

confidence: 99%

The Human Herpes Virus 8-Encoded Chemokine Receptor Is Required for Angioproliferation in a Murine Model of Kaposi’s Sarcoma

Jensen

Manfra²,

Grisotto³

et al. 2005

The Journal of Immunology

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“…In the case of Kaposi's sarcoma, ORF74 is believed to be one of the primary oncogenes because transgenic mice expressing this receptor develop lesions closely resembling those of the human disease (17,18) in agreement with its proliferative capability in vitro (11). Importantly, the constitutive activity of ORF74 was shown to be essential for this process because mice expressing non-constitutively active ORF74 mutants had a marked lower index of tumorigenesis (19). In support of this observation, cells expressing non-constitutively active US28 mutants were less efficient in promoting tumor development compared with WT counterparts when injected into nude mice (20).…”

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confidence: 99%

“…It is interesting to note that the expression of EBI2 is massively up-regulated under this program, as seen both in vitro (1,46) and in EBV-positive PTLD samples (23). Given the association between 7TM receptor constitutive activity and tumorigenesis (11,19), it is tempting to speculate that EBI2 in PTLD and other latency III-associated diseases could function as an oncogene. This is substantiated by the observation that overexpression of EBI2 profoundly potentiates the antibody-induced proliferation rate of B cells, whereas EBI2 deficiency has the opposite effect (Figs.…”

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confidence: 99%

Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

Benned-Jensen

Smethurst

Holst

et al. 2011

Journal of Biological Chemistry

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The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5-3-O-(thio)-triphosphate (GTP␥S) binding assays, the potency of this compound was 2.6 -53.6 nM, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTP␥S binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe 111 at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC 50 . In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.The Epstein-Barr virus (EBV) induced receptor 2 (EBI2; also known as GPR183) is an orphan member of the 7TM 2 receptor family A. EBI2 is up-regulated up to 200-fold in B cells following EBV infection (1). In agreement with an expression pattern primarily restricted to immunological cell types (1, 2), it was recently demonstrated that EBI2 orchestrates the positioning of B cells in the follicle (3, 4). Specifically, expression of EBI2 allowed activated B cells to translocate to the outer follicular regions; in contrast, the absence of EBI2 permitted the cells to enter the germinal centers. Thus, EBI2 mediates the correct localization of B cells during humoral immune responses. To date, no endogenous ligand for EBI2 has been identified. Given that this receptor does not have a close homolog, the chemical nature of such a ligand is difficult to infer. Accordingly, EBI2 has been placed in varying 7TM receptor subgroups by different phylogenetic analyses as being a target of peptide (1, 5) or lipid ligands (6). Nevertheless, many aspects of EBI2 signaling have been revealed. Thus, we have previously demonstrated that EBI2 is constitutively active through G␣ i (but not G␣ q or G␣ s ) and serum response element (but not NFAT or NFB...

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“…Nevertheless, high constitutive activity is indisputably a hallmark of many virally encoded 7TM receptors such as ORF74 from human herpes virus 8 and US28 from human cytomegalovirus (5). The virus has optimized, for example, ORF74 to signal constitutively with ϳ50% efficacy and to be modulated positively by endogenous angiogenic chemokines and negatively by endogenous angiostatic or modulatory chemokines (6,7).…”

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confidence: 99%

Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family

Holst

Holliday

Bach

et al. 2004

Journal of Biological Chemistry

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305

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Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G q , phospholipase C pathway was ϳ50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G 12/13 and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.

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Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

Cited by 100 publications

References 32 publications

The Human Herpes Virus 8-Encoded Chemokine Receptor Is Required for Angioproliferation in a Murine Model of Kaposi’s Sarcoma

The Human Herpes Virus 8-Encoded Chemokine Receptor Is Required for Angioproliferation in a Murine Model of Kaposi’s Sarcoma

Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family

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