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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 mediated by metalloproteases.(A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. 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-g003: CAR shedding is mediated by metalloproteases.(A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.

Mentions: To determine which protease family mediates the ECD shedding of CAR, various protease inhibitors were used in conjunction with PMA treatment of U87 CAR cells. The broad-spectrum metalloprotease inhibitors O-phenathroline and TAPI-1 inhibited PMA-stimulated CAR ECD shedding, but the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin did not (Figure 3A). GM6001, another broad-spectrum metalloprotease inhibitor, inhibited PMA-stimulated CAR ECD shedding (Figure 3B) as well as constitutive shedding (data not shown). Thus, metalloproteases, and not other classes of proteases, are required for constitutive and PMA-induced ECD shedding of CAR.


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 mediated by metalloproteases.(A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. 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-g003: CAR shedding is mediated by metalloproteases.(A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.
Mentions: To determine which protease family mediates the ECD shedding of CAR, various protease inhibitors were used in conjunction with PMA treatment of U87 CAR cells. The broad-spectrum metalloprotease inhibitors O-phenathroline and TAPI-1 inhibited PMA-stimulated CAR ECD shedding, but the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin did not (Figure 3A). GM6001, another broad-spectrum metalloprotease inhibitor, inhibited PMA-stimulated CAR ECD shedding (Figure 3B) as well as constitutive shedding (data not shown). Thus, metalloproteases, and not other classes of proteases, are required for constitutive and PMA-induced ECD shedding of CAR.

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