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Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment.

Mazot P, Cazes A, Dingli F, Degoutin J, Irinopoulou T, Boutterin MC, Lombard B, Loew D, Hallberg B, Palmer RH, Delattre O, Janoueix-Lerosey I, Vigny M - PLoS ONE (2012)

Bottom Line: We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT).We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation.This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie, Paris, France.

ABSTRACT
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

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Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor.A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.
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pone-0033581-g003: Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor.A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.

Mentions: We first determined whether agonist mAb could trigger ALK internalization. The low level of ALK expression in neuroblastoma cell lines did not allow us to follow internalization by immunofluorescence. We thus employed transiently transfected ALKWT in CHO cells. To investigate the internalization of ALK primarily located at the membrane before treatment, we incubated living cells with agonist mAb 46 for 10 min at 4°C in order to block endocytosis. We then chased cells at 37°C and observed the resultant, in order to allow receptor trafficking. Using confocal microscopy we were able to detect mAb 46 bound to the ALK receptor. As expected, after incubation at 4°C we could only detect the mAb∶ALK receptor complex at the cell surface. After 15′ of incubation at 37°C ALK appeared intracellularly, and after 3 h the staining was essentially intracellular (Fig. 3A).


Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment.

Mazot P, Cazes A, Dingli F, Degoutin J, Irinopoulou T, Boutterin MC, Lombard B, Loew D, Hallberg B, Palmer RH, Delattre O, Janoueix-Lerosey I, Vigny M - PLoS ONE (2012)

Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor.A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316580&req=5

pone-0033581-g003: Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor.A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.
Mentions: We first determined whether agonist mAb could trigger ALK internalization. The low level of ALK expression in neuroblastoma cell lines did not allow us to follow internalization by immunofluorescence. We thus employed transiently transfected ALKWT in CHO cells. To investigate the internalization of ALK primarily located at the membrane before treatment, we incubated living cells with agonist mAb 46 for 10 min at 4°C in order to block endocytosis. We then chased cells at 37°C and observed the resultant, in order to allow receptor trafficking. Using confocal microscopy we were able to detect mAb 46 bound to the ALK receptor. As expected, after incubation at 4°C we could only detect the mAb∶ALK receptor complex at the cell surface. After 15′ of incubation at 37°C ALK appeared intracellularly, and after 3 h the staining was essentially intracellular (Fig. 3A).

Bottom Line: We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT).We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation.This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie, Paris, France.

ABSTRACT
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

Show MeSH
Related in: MedlinePlus