Henry Vischer

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Organization: Vrije Universiteit Amsterdam , Belgium

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Title: (PhD)

Chemicals
References

Identification and profiling of CXCR3CXCR4 chemokine receptor heteromer complexes

Co-reporter:AO Watts;MMH van Lipzig;WC Jaeger;RM Seeber;M van Zwam;J Vinet;MMC van der Lee;M Siderius;GJR Zaman;HWGM Boddeke;MJ Smit;KDG Pfleger;R Leurs;HF Vischer

British Journal of Pharmacology 2013 Volume 168( Issue 7) pp:1662-1674

Publication Date(Web):

DOI:10.1111/bph.12064

Background and Purpose

The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.

Experimental Approach

CXCR3–CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity.

Key Results

We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific β-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation.

Conclusions and Implications

This study indicates that heteromeric CXCR3–CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.

The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R

Co-reporter:Sabrina M. de Munnik, Rosan van der Lee, Daniëlle M. Velders, Jody van Offenbeek, Laura Smits-de Vries, Rob Leurs, Martine J. Smit, Henry F. Vischer

Cellular Signalling (June 2016) Volume 28(Issue 6) pp:595-605

Publication Date(Web):1 June 2016

DOI:10.1016/j.cellsig.2016.02.017

•The viral chemokine receptor ORF74 is unable to transactivate IGF-1R•IGF-1 does not directly activate PLC in HEK293T cells endogenously expressing IGF-1R•IGF-1 activates PLC in an ORF74-dependent manner•IGF-1 activates PLC independently of Src, PI3K, ORF74 ligands and ORF74 tyrosine residues•IGF-1-induced PLC activation is dependent on IGF-1R and the basal activity of ORF74Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival.In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.Download high-res image (292KB)Download full-size image