Transmembrane chemokines act as receptors in a novel mechanism termed inverse signaling
- University of Kiel, Germany
- Institute of Zoology, Germany
- RWTH Aachen University, Germany
- University Medical Center Schleswig-Holstein, Germany
Figures
Figure 1
Expression of transmembrane chemokines and their known receptors in various cell types.
Top: As determined by qRT-PCR, the transmembrane chemokines CXCL16 and CX3CL1 are highly transcribed in many human tumor cell lines including glioma (U118, U343, T98G, A172, A764), colon carcinoma …
Figure 2 with 2 supplements
Signal transduction in receptor-negative (CXCR6-, CX3CR1-) tumor cells after stimulation with soluble chemokines (1 nM s-CXCL16 or s-CX3CL1).
A) As shown by Western blots after SDS-PAGE separation, receptor-negative but hence responsive cell lines like the glioma cell lines U343, A764, T98G and primary glioma cultures from different …
Figure 2—figure supplement 1
Biological replicates of western blot experiments showing time- and dose-dependent activation of ERK1/2 upon stimulation with s-chemokines in responsive glioma cell lines (compare Figure 2A)
https://doi.org/10.7554/eLife.10820.005
Figure 2—figure supplement 2
Biological replicates of western blot experiments showing stimulations of non-responsive LOX melanoma cells with s-chemokines (compare Figure 2C)
https://doi.org/10.7554/eLife.10820.006
Figure 3 with 1 supplement
Biological effects after stimulation of receptor-negative (CXCR6-, CX3CR1-) tumor cells with soluble chemokines (1 nM s-CXCL16 or s-CX3CL1).
Top: Soluble chemokines (1 nM) enhance proliferation of U343 and A764 glioma cells expressing the transmembrane counterparts, but not of LOX melanoma cells that are tm-chemokine negative. …
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Figure 3—source data 1
Biological replicates of the scratch assay shown in Figure 3.
- https://doi.org/10.7554/eLife.10820.008
Figure 3—figure supplement 1
Only slight activation and effects upon s-chemokine stimulation of tm-chemokine low expressing MCF-7 cells.
Top: MCF-7 breast carcinoma cells were stimulated with 1 nM s-CXCL16 or s-CX3CL1 for 20 min and subjected to Western blot on ERK1/2 phosphorylation. In comparison to positive controls (EGF and …
Figure 4 with 1 supplement
Inhibition experiments and transcription analysis exclude the involvement of other chemokine receptors.
(A) Pertussis toxin (PTX, 200 ng/ml) inhibiting Gi/o-signaling of classical chemokine receptors has no effect on s-chemokine-mediated phosphorylation of kinases in U343 or A764 cells. However, in …
Figure 4—figure supplement 1
Biological replicates of western blot experiments with s-chemokines and Pertussis toxin (compare Figure 4A).
https://doi.org/10.7554/eLife.10820.011
Figure 5
Binding of soluble chemokines to corresponding tm-chemokines on cell surfaces.
(A) Fluorescent soluble chemokines, either labeled directly (cyanine3, Cy3) or indirectly via biotin-label to fluorescent (strept)avidin (Alexa-Fluor 488 or fluorescein-isothiocyanate, FITC), bind …
Figure 6 with 1 supplement
Non-responsive cells can be transformed to be responsive to s-chemokine stimulation by transfection with tm-chemokines.
(A) LOX melanoma cells were stably transfected with tm-chemokines. Transfection efficiency was controlled by quantitative RT-PCR and immunocytochemistry (n = 3 biological replicates as indicated by …
Figure 6—figure supplement 1
Biological replicates of western blot experiments with transfected LOX melanoma cells (compare Figure 6).
https://doi.org/10.7554/eLife.10820.014
Figure 7
Stimulation with s-chemokines can mediate rescue from chemically-induced cell death in tm-chemokine-transfected LOX melanoma cells.
LOX melanoma cells stably expressing tm-CXCL16 or tm-CX3CL1 (or mock transfected LOX cells) were treated for 18 hr with 0.1 µg/ml Camptothecin (inhibitor of topoisomerase I) to induce cell death. …
Figure 8 with 1 supplement
Silencing of tm-chemokine expression abolishes the s-chemokine mediated activation in responsive glioma cell lines and primary cultures.
Cell lines and primary cells were transfected with CXCL16 or CX3CL1 specific RNAi (or non-specific control RNAi), left for recovery for 36 hr and stimulated with 1nM CXCL16 or CX3CL1 for 15 min. …
Figure 8—figure supplement 1
Biological replicates of western blot experiments from siRNA knockdown in T98G glioma cells (compare Figure 8).
https://doi.org/10.7554/eLife.10820.017
Figure 9 with 1 supplement
Signal transduction in tm-chemokine expressing LOX cells upon stimulation with specific antibodies (0.1 µg/ml), but not monovalent F(ab) fragments.
(A) As shown by Western blot after SDS-PAGE, stimulation of tm-CXCL16 or tm-CX3CL1 transfected LOX cells for 20 min with antibodies against the corresponding chemokine domains (0.1 µg/ml), yields a …
Figure 9—figure supplement 1
Biological replicates of western blot experiments of stimulations with chemokine-specific antibodies (compare Figure 9).
https://doi.org/10.7554/eLife.10820.019Tables
Table 1
Sequences of putative intracellular domains from transmembrane chemokines.
| CX3CL1 |
Homo sapiens (Human) | -QSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV |
| Nomascus leucogenys (Northern white-cheeked gibbon) | -QSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV |
| Macaca mulatta (Rhesus macaque) | -QSLQGCP RKMAGEMVEGLRYIPRSCGS NSYVLVPV |
| Bos taurus (Bovine) | -QRLQSCPHKMVGDVVEGICYVPRSCGSNSYVLVPV |
| Canis familiaris (Dog) | -YQSLQGCSR KMAGDMVEGLRYVPRSCGSN SYVLVPV |
| Oryctolagus cuniculus (Rabbit) | -Q SLQGCPRKMAGEMVEGLRYV PRSCGANSYVLVPV |
| Cavia porcellus (Guinea pig) | -QSLQGCPRK MAGEMVEGLRYVPRSCGSNSYVLVPV |
| Rattus norwegicus (Rat) | -QS LQGCPRKMAG EMVEGLRYVP RSCGSNSYVLVPV |
| Mus musculus (Mouse) | -QSLQGCPRKM AGEMVEGLRYVPRSCGSNSYVLVPV |
| Monodelphis domestica (Gray short-tailed opossum) | -QSLQSCPRRMAGEVVEGLRYIPRSCGSNSYVLVPV |
| Sarcophilus harrisii (Tasman devil) | -QSLQSCPRRMAGEVVEGLRYIPRSCGSNSYVLVPV |
| Ornithorhynchus anatinus (Duckbill platypus) | -QSLQSCPRRMAGEVVEGLRYIPRSCGSNSYVLVPV |
| CXCL16 | |
Homo sapiens (Human) | -CKRRRGQSPQSSPD PVHYIPVAP DSNT |
| Gorilla gorilla gorilla (Lowland gorilla) | -CKRRRGQSPQSSPGLPVHYIPVAPDSNT |
| Nomascus leucogenys (Northern white-cheeked gibbon) | -CKR RRGQSPQSSPDLQFHYIPVA PDSNT |
| Macaca mulatta (Rhesus macaque) | -CKRRGQSPQSSPDLQLHYIPVASDSNT |
| Sus scrofa (Pig) | -CKKRQEQSRQYPPDPQLHYVPVASNINT |
| Loxodonta africana (African elephant) | -CKRRREQSRLYYPDLQFHYKPVA PDS |
| Bos taurus (Bovine) | -C KRRKNQLLQHPPDLAASLYT CSRRTRAENGTL |
| Equus caballus (Horse) | -CKKREKTLRPSPDLQAHYERVAPD |
| Canis familiaris (Dog) | -CKRREQSLQHPPDLQLHYTPVASDSNV |
| Oryctolagus cuniculus (Rabbit) | -CKRRRGRSPKYSSGKP |
| Rattus norwegicus (Rat) | -CNRRVTRQSSSGLQLCYTPV EPRPQGL |
| Mus musculus (Mouse) | -CNRRATQQNSAGLQLWYTPVEPRP |
| Myotis lucifugus (Little brown bat) | -CKRRSKQSPQYSPDLQLQCIPVASYSNS |
| Ornithorhynchus anatinus (Duckbill platypus) | -CRRRGAPRNEMLYPQRPKGTSITVQANSPT |
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Data from: Uniprot (http://www.uniprot.org/)
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Putative intracellular domains are depicted by homology to the published putative human sequences.
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-SXXS- motifs, -SXXT- motifs, -SXS- motifs; -SXPV/R- SH2-binding site
