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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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CAR shedding is stimulated by the calcium ionophore ionomycin and the phorbol ester PMA.(A) U87 CAR cells were treated with the calcium ionophore ionomycin at the indicated concentrations for 30 minutes. Ionomycin treatment stimulated the ECD shedding of CAR. (B) PMA treatment (25 ng/ml) of U87 CAR cells did not trigger CAR ECD shedding within one hour. At this concentration, 4 hours of PMA treatment led to robust CAR ECD shedding, but remained lower than constitutive shedding over 16 hours. Volumes of conditioned media loaded on SDS-PAGE were adjusted according to lysate protein concentrations. (C) The protein kinase C (PKC) inhibitor Gö 6983 decreased PMA-stimulated shedding of CAR ECD into conditioned media in a dose-dependent manner. PMA was used at a final concentration of 1 μM, and Gö 6983 at the following concentrations: + = 1 nM; ++ = 10 nM, and +++ = 100 nM. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.
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pone-0073296-g002: CAR shedding is stimulated by the calcium ionophore ionomycin and the phorbol ester PMA.(A) U87 CAR cells were treated with the calcium ionophore ionomycin at the indicated concentrations for 30 minutes. Ionomycin treatment stimulated the ECD shedding of CAR. (B) PMA treatment (25 ng/ml) of U87 CAR cells did not trigger CAR ECD shedding within one hour. At this concentration, 4 hours of PMA treatment led to robust CAR ECD shedding, but remained lower than constitutive shedding over 16 hours. Volumes of conditioned media loaded on SDS-PAGE were adjusted according to lysate protein concentrations. (C) The protein kinase C (PKC) inhibitor Gö 6983 decreased PMA-stimulated shedding of CAR ECD into conditioned media in a dose-dependent manner. PMA was used at a final concentration of 1 μM, and Gö 6983 at the following concentrations: + = 1 nM; ++ = 10 nM, and +++ = 100 nM. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.

Mentions: ECD shedding of cell surface proteins can be controlled by cell signaling pathways, with regulated stimulation of ECD shedding occurring within a relatively short period of time compared to constitutive shedding. For example, the calcium ionophore ionomycin can induce rapid cell surface protein shedding [36] through stimulation of ADAM10 [37,38]. As seen in Figure 2A, treatment of U87 CAR cells with ionomycin for 30 minutes upregulated CAR ECD shedding. Phorbol esters such as PMA activate the PKC pathway [39] and also induce shedding of cell surface proteins [40]. Shedding of substrates via treatment with low concentrations of PMA for 1 hour or less is generally mediated by ADAM17 [37,41]. In the case of U87 CAR cells treated with 25 ng/ml of PMA, substantial levels of CAR ECD were detected from conditioned media only after 2 hours of treatment, suggesting that ADAM17 may not be involved in CAR ECD shedding (Figure 2B). PMA-induced shedding of CAR ECD was inhibited by the PKC inhibitor Gö 6983 in a dose-dependent manner (Figure 2C). Thus, CAR shedding is regulated by signaling pathways.


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

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

CAR shedding is stimulated by the calcium ionophore ionomycin and the phorbol ester PMA.(A) U87 CAR cells were treated with the calcium ionophore ionomycin at the indicated concentrations for 30 minutes. Ionomycin treatment stimulated the ECD shedding of CAR. (B) PMA treatment (25 ng/ml) of U87 CAR cells did not trigger CAR ECD shedding within one hour. At this concentration, 4 hours of PMA treatment led to robust CAR ECD shedding, but remained lower than constitutive shedding over 16 hours. Volumes of conditioned media loaded on SDS-PAGE were adjusted according to lysate protein concentrations. (C) The protein kinase C (PKC) inhibitor Gö 6983 decreased PMA-stimulated shedding of CAR ECD into conditioned media in a dose-dependent manner. PMA was used at a final concentration of 1 μM, and Gö 6983 at the following concentrations: + = 1 nM; ++ = 10 nM, and +++ = 100 nM. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756012&req=5

pone-0073296-g002: CAR shedding is stimulated by the calcium ionophore ionomycin and the phorbol ester PMA.(A) U87 CAR cells were treated with the calcium ionophore ionomycin at the indicated concentrations for 30 minutes. Ionomycin treatment stimulated the ECD shedding of CAR. (B) PMA treatment (25 ng/ml) of U87 CAR cells did not trigger CAR ECD shedding within one hour. At this concentration, 4 hours of PMA treatment led to robust CAR ECD shedding, but remained lower than constitutive shedding over 16 hours. Volumes of conditioned media loaded on SDS-PAGE were adjusted according to lysate protein concentrations. (C) The protein kinase C (PKC) inhibitor Gö 6983 decreased PMA-stimulated shedding of CAR ECD into conditioned media in a dose-dependent manner. PMA was used at a final concentration of 1 μM, and Gö 6983 at the following concentrations: + = 1 nM; ++ = 10 nM, and +++ = 100 nM. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.
Mentions: ECD shedding of cell surface proteins can be controlled by cell signaling pathways, with regulated stimulation of ECD shedding occurring within a relatively short period of time compared to constitutive shedding. For example, the calcium ionophore ionomycin can induce rapid cell surface protein shedding [36] through stimulation of ADAM10 [37,38]. As seen in Figure 2A, treatment of U87 CAR cells with ionomycin for 30 minutes upregulated CAR ECD shedding. Phorbol esters such as PMA activate the PKC pathway [39] and also induce shedding of cell surface proteins [40]. Shedding of substrates via treatment with low concentrations of PMA for 1 hour or less is generally mediated by ADAM17 [37,41]. In the case of U87 CAR cells treated with 25 ng/ml of PMA, substantial levels of CAR ECD were detected from conditioned media only after 2 hours of treatment, suggesting that ADAM17 may not be involved in CAR ECD shedding (Figure 2B). PMA-induced shedding of CAR ECD was inhibited by the PKC inhibitor Gö 6983 in a dose-dependent manner (Figure 2C). Thus, CAR shedding is regulated by signaling pathways.

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