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Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

Schmieg N, Thomas C, Yabe A, Lynch DS, Iglesias T, Chakravarty P, Schiavo G - PLoS ONE (2015)

Bottom Line: The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33.Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript.Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropathobiology Laboratory, Sobell Department of Motor Neuroscience & Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, United Kingdom; The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.

ABSTRACT
Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

No MeSH data available.


Related in: MedlinePlus

Distinct Kidins220 splice isoforms display specific cellular localisations.(A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.
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pone.0129944.g007: Distinct Kidins220 splice isoforms display specific cellular localisations.(A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.

Mentions: NGF treatment is known to change the cellular distribution of Kidins220 from the plasma membrane to the neurite tips in differentiated PC12 cells [34], a process which relies on the recruitment of kinesin-1 to the carboxy-terminal domain of Kidins220 and is required for physiological TrkA signalling [31]. To determine potential differences in the localisation of Kidins220 isoforms in PC12 cells, we selected isoform m6 and m6/C2 (Fig 7A), which are endogenously expressed in these cells (data not shown). We transfected HA-tagged constructs of isoforms m6, isoform m6/C2 and pLVX TetON vector only (control) into undifferentiated PC12 cells, which were then treated with NGF for 48 h (Fig 7) and into HEK cells (S6 Fig). Cells were fixed and stained for exon 32 (including endogenous and exogenous Kidins220 isoforms m6) using an antibody directed against the carboxy-terminus of Kidins220 (GSC16) or total Kidins220 level (KNA) and for the exogenous variants using an anti-HA antibody (Fig 7B and S6 Fig, respectively).


Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

Schmieg N, Thomas C, Yabe A, Lynch DS, Iglesias T, Chakravarty P, Schiavo G - PLoS ONE (2015)

Distinct Kidins220 splice isoforms display specific cellular localisations.(A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470590&req=5

pone.0129944.g007: Distinct Kidins220 splice isoforms display specific cellular localisations.(A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.
Mentions: NGF treatment is known to change the cellular distribution of Kidins220 from the plasma membrane to the neurite tips in differentiated PC12 cells [34], a process which relies on the recruitment of kinesin-1 to the carboxy-terminal domain of Kidins220 and is required for physiological TrkA signalling [31]. To determine potential differences in the localisation of Kidins220 isoforms in PC12 cells, we selected isoform m6 and m6/C2 (Fig 7A), which are endogenously expressed in these cells (data not shown). We transfected HA-tagged constructs of isoforms m6, isoform m6/C2 and pLVX TetON vector only (control) into undifferentiated PC12 cells, which were then treated with NGF for 48 h (Fig 7) and into HEK cells (S6 Fig). Cells were fixed and stained for exon 32 (including endogenous and exogenous Kidins220 isoforms m6) using an antibody directed against the carboxy-terminus of Kidins220 (GSC16) or total Kidins220 level (KNA) and for the exogenous variants using an anti-HA antibody (Fig 7B and S6 Fig, respectively).

Bottom Line: The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33.Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript.Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropathobiology Laboratory, Sobell Department of Motor Neuroscience & Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, United Kingdom; The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.

ABSTRACT
Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

No MeSH data available.


Related in: MedlinePlus