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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

ADAM10 is involved in CAR ECD shedding.(A) U251N cells stably expressing CAR were transfected with empty plasmid (mock) or ADAM10 plasmid. 24 hours after transfection, cells were washed and incubated in opti-MEM for 24 hours, and conditioned media and cell lysates were analyzed by Western blots using anti-CAR N-terminus antibody (2239). Overexpression of ADAM10 increased constitutive CAR ECD shedding. (B) U87 CAR cells were treated with 10 μM purified ADAM10 prodomain (versus an equivalent volume of buffer as a control), and conditioned media and cell lysates were collected as previously described. A Western blot for CAR extracellular domain (2240 antibody) shows that the prodomain of ADAM10, which inhibits ADAM10 activity, decreased CAR ECD shedding.
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pone-0073296-g004: ADAM10 is involved in CAR ECD shedding.(A) U251N cells stably expressing CAR were transfected with empty plasmid (mock) or ADAM10 plasmid. 24 hours after transfection, cells were washed and incubated in opti-MEM for 24 hours, and conditioned media and cell lysates were analyzed by Western blots using anti-CAR N-terminus antibody (2239). Overexpression of ADAM10 increased constitutive CAR ECD shedding. (B) U87 CAR cells were treated with 10 μM purified ADAM10 prodomain (versus an equivalent volume of buffer as a control), and conditioned media and cell lysates were collected as previously described. A Western blot for CAR extracellular domain (2240 antibody) shows that the prodomain of ADAM10, which inhibits ADAM10 activity, decreased CAR ECD shedding.

Mentions: Transient overexpression of wild-type ADAM10 in human glioma U251N cells stably expressing CAR (U251N CAR) increased shedding of CAR ECD (Figure 4A). Purified recombinant prodomain of ADAM10 inhibits ADAM10 activity when added to cell culture media [43]. Addition of ADAM10 prodomain markedly decreased constitutive shedding of CAR (Figure 4B). These results suggest that the metalloprotease ADAM10 is involved in CAR ECD shedding.


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

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

ADAM10 is involved in CAR ECD shedding.(A) U251N cells stably expressing CAR were transfected with empty plasmid (mock) or ADAM10 plasmid. 24 hours after transfection, cells were washed and incubated in opti-MEM for 24 hours, and conditioned media and cell lysates were analyzed by Western blots using anti-CAR N-terminus antibody (2239). Overexpression of ADAM10 increased constitutive CAR ECD shedding. (B) U87 CAR cells were treated with 10 μM purified ADAM10 prodomain (versus an equivalent volume of buffer as a control), and conditioned media and cell lysates were collected as previously described. A Western blot for CAR extracellular domain (2240 antibody) shows that the prodomain of ADAM10, which inhibits ADAM10 activity, decreased CAR ECD shedding.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073296-g004: ADAM10 is involved in CAR ECD shedding.(A) U251N cells stably expressing CAR were transfected with empty plasmid (mock) or ADAM10 plasmid. 24 hours after transfection, cells were washed and incubated in opti-MEM for 24 hours, and conditioned media and cell lysates were analyzed by Western blots using anti-CAR N-terminus antibody (2239). Overexpression of ADAM10 increased constitutive CAR ECD shedding. (B) U87 CAR cells were treated with 10 μM purified ADAM10 prodomain (versus an equivalent volume of buffer as a control), and conditioned media and cell lysates were collected as previously described. A Western blot for CAR extracellular domain (2240 antibody) shows that the prodomain of ADAM10, which inhibits ADAM10 activity, decreased CAR ECD shedding.
Mentions: Transient overexpression of wild-type ADAM10 in human glioma U251N cells stably expressing CAR (U251N CAR) increased shedding of CAR ECD (Figure 4A). Purified recombinant prodomain of ADAM10 inhibits ADAM10 activity when added to cell culture media [43]. Addition of ADAM10 prodomain markedly decreased constitutive shedding of CAR (Figure 4B). These results suggest that the metalloprotease ADAM10 is involved in CAR ECD shedding.

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