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The Coxsackievirus and Adenovirus Receptor (CAR) undergoes ectodomain shedding and regulated intramembrane proteolysis (RIP).

Houri N, Huang KC, Nalbantoglu J - PLoS ONE (2013)

Bottom Line: CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus.Ectodomain shedding is a prerequisite for RIP of CAR.Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates' ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.

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Related in: MedlinePlus

CAR’s intracellular domain (ICD) enters the nucleus.Immunofluorescence and confocal microscopy images showing the presence of CAR ICD in nuclei of U87-MG cells. U87-MG cells were transiently transfected with empty pcDNA3.1 V5/His plasmid, full-length CAR-V5 plasmid or with CAR ICD-V5 plasmid. Immunofluorescence staining was performed 24-48 hours post-transfection using anti-V5 tag antibody and Alexa Fluor 555 secondary antibody (red). Nuclei were stained with DRAQ5 (blue). Images were acquired with a confocal microscope (63x oil objective). Images are representative of at least 3 independent experiments. Scale bars: 5 µm.
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pone-0073296-g010: CAR’s intracellular domain (ICD) enters the nucleus.Immunofluorescence and confocal microscopy images showing the presence of CAR ICD in nuclei of U87-MG cells. U87-MG cells were transiently transfected with empty pcDNA3.1 V5/His plasmid, full-length CAR-V5 plasmid or with CAR ICD-V5 plasmid. Immunofluorescence staining was performed 24-48 hours post-transfection using anti-V5 tag antibody and Alexa Fluor 555 secondary antibody (red). Nuclei were stained with DRAQ5 (blue). Images were acquired with a confocal microscope (63x oil objective). Images are representative of at least 3 independent experiments. Scale bars: 5 µm.

Mentions: Finally, as RIP of some cell surface proteins produces intracellular fragments that enter the nucleus, we investigated if that is the case for CAR. Nuclear immunoreactivity was observed with transient expression of full-length CAR-V5 in 293 cells (data not shown); however, the appearance of these speckles was rare. We generated a construct expressing the intracellular domain (amino acids 261-365 of murine CAR isoform 1) tagged at the C-terminus with V5, named CAR ICD-V5. U87-MG cells were transiently transfected with empty plasmid, full-length CAR-V5 or CAR ICD-V5, and immunofluorescence experiments were performed. Images were acquired using a confocal microscope. The ICD was readily detected in nuclei of U87 cells (Figure 10 and Figure S7). Similar results were obtained from experiments using 293A cells (data not shown). Thus, the intracellular domain of CAR is capable of nuclear entry.


The Coxsackievirus and Adenovirus Receptor (CAR) undergoes ectodomain shedding and regulated intramembrane proteolysis (RIP).

Houri N, Huang KC, Nalbantoglu J - PLoS ONE (2013)

CAR’s intracellular domain (ICD) enters the nucleus.Immunofluorescence and confocal microscopy images showing the presence of CAR ICD in nuclei of U87-MG cells. U87-MG cells were transiently transfected with empty pcDNA3.1 V5/His plasmid, full-length CAR-V5 plasmid or with CAR ICD-V5 plasmid. Immunofluorescence staining was performed 24-48 hours post-transfection using anti-V5 tag antibody and Alexa Fluor 555 secondary antibody (red). Nuclei were stained with DRAQ5 (blue). Images were acquired with a confocal microscope (63x oil objective). Images are representative of at least 3 independent experiments. Scale bars: 5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756012&req=5

pone-0073296-g010: CAR’s intracellular domain (ICD) enters the nucleus.Immunofluorescence and confocal microscopy images showing the presence of CAR ICD in nuclei of U87-MG cells. U87-MG cells were transiently transfected with empty pcDNA3.1 V5/His plasmid, full-length CAR-V5 plasmid or with CAR ICD-V5 plasmid. Immunofluorescence staining was performed 24-48 hours post-transfection using anti-V5 tag antibody and Alexa Fluor 555 secondary antibody (red). Nuclei were stained with DRAQ5 (blue). Images were acquired with a confocal microscope (63x oil objective). Images are representative of at least 3 independent experiments. Scale bars: 5 µm.
Mentions: Finally, as RIP of some cell surface proteins produces intracellular fragments that enter the nucleus, we investigated if that is the case for CAR. Nuclear immunoreactivity was observed with transient expression of full-length CAR-V5 in 293 cells (data not shown); however, the appearance of these speckles was rare. We generated a construct expressing the intracellular domain (amino acids 261-365 of murine CAR isoform 1) tagged at the C-terminus with V5, named CAR ICD-V5. U87-MG cells were transiently transfected with empty plasmid, full-length CAR-V5 or CAR ICD-V5, and immunofluorescence experiments were performed. Images were acquired using a confocal microscope. The ICD was readily detected in nuclei of U87 cells (Figure 10 and Figure S7). Similar results were obtained from experiments using 293A cells (data not shown). Thus, the intracellular domain of CAR is capable of nuclear entry.

Bottom Line: CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus.Ectodomain shedding is a prerequisite for RIP of CAR.Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates' ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.

Show MeSH
Related in: MedlinePlus