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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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shRNA knockdown of ADAM10 decreases constitutive, PMA-mediated and ionomycin-mediated CAR ECD shedding.(A) A Western blot for ADAM10 using cell lysates of U87 CAR cells containing either control shRNA or ADAM10 (#6676) shRNA, in biological duplicates. Equal amounts of proteins were loaded on SDS-PAGE gel. Anti-GAPDH antibody was used as a loading control. Quantification of mean ADAM10 band intensities normalized over GAPDH revealed a decrease of approximately 60% with anti-ADAM10 shRNA compared to control shRNA. (B) A constitutive shedding experiment was performed using U87 CAR stable cell lines containing either control shRNA or anti-ADAM10 shRNA (#6676). Conditioned media and cell lysates were collected after 24 hours of incubation of cells in opti-MEM, and Western blotting was done using the anti-CAR N-terminus antibody 2239. With anti-ADAM10 shRNA, there was a significant decrease (40%) in the levels of shed CAR. Results from 4 independent experiments performed in duplicates were quantified (unpaired t-test; p=0.0004 (***)). (C) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6676) were treated with 1 µM PMA. Conditioned media and cell lysates were collected after 3 hours, and Western blots were performed using the anti-CAR N-terminus antibody 2239. With shRNA knockdown of ADAM10, there was a significant decrease of 41% in levels of shed CAR compared to control shRNA. Results from 3 independent experiments performed in duplicates were quantified (one-way ANOVA with Tukey’s multiple comparison test; *** = p < 0.001). (D) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6675) were treated with 1.5 µM ionomycin (vs. DMSO vehicle). Conditioned media and cell lysates were collected after 30 minutes of treatment, and Western blots were performed using the anti-CAR N-terminus antibody 2240. With shRNA knockdown of ADAM10, there was a significant decrease of 52% in levels of shed CAR with ionomycin treatment compared to control shRNA. Results from 3 independent experiments (n=3 per group) were quantified (one-way ANOVA with Bonferroni’s multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).
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pone-0073296-g005: shRNA knockdown of ADAM10 decreases constitutive, PMA-mediated and ionomycin-mediated CAR ECD shedding.(A) A Western blot for ADAM10 using cell lysates of U87 CAR cells containing either control shRNA or ADAM10 (#6676) shRNA, in biological duplicates. Equal amounts of proteins were loaded on SDS-PAGE gel. Anti-GAPDH antibody was used as a loading control. Quantification of mean ADAM10 band intensities normalized over GAPDH revealed a decrease of approximately 60% with anti-ADAM10 shRNA compared to control shRNA. (B) A constitutive shedding experiment was performed using U87 CAR stable cell lines containing either control shRNA or anti-ADAM10 shRNA (#6676). Conditioned media and cell lysates were collected after 24 hours of incubation of cells in opti-MEM, and Western blotting was done using the anti-CAR N-terminus antibody 2239. With anti-ADAM10 shRNA, there was a significant decrease (40%) in the levels of shed CAR. Results from 4 independent experiments performed in duplicates were quantified (unpaired t-test; p=0.0004 (***)). (C) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6676) were treated with 1 µM PMA. Conditioned media and cell lysates were collected after 3 hours, and Western blots were performed using the anti-CAR N-terminus antibody 2239. With shRNA knockdown of ADAM10, there was a significant decrease of 41% in levels of shed CAR compared to control shRNA. Results from 3 independent experiments performed in duplicates were quantified (one-way ANOVA with Tukey’s multiple comparison test; *** = p < 0.001). (D) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6675) were treated with 1.5 µM ionomycin (vs. DMSO vehicle). Conditioned media and cell lysates were collected after 30 minutes of treatment, and Western blots were performed using the anti-CAR N-terminus antibody 2240. With shRNA knockdown of ADAM10, there was a significant decrease of 52% in levels of shed CAR with ionomycin treatment compared to control shRNA. Results from 3 independent experiments (n=3 per group) were quantified (one-way ANOVA with Bonferroni’s multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Mentions: To further confirm ADAM10’s role in CAR shedding, we used shRNA to knock down ADAM10 expression in U87 CAR cells. Stable cell lines were generated for anti-eGFP shRNA (used as control) or anti-ADAM10 shRNA (sequences #6675 or #6676). The shRNA sequences targeting ADAM10 successfully knocked down ADAM10 mRNA levels (Figure S3) and, subsequently, ADAM10 protein levels (Figure 5A). The shRNA stable cell lines were then used to study constitutive and regulated CAR ECD shedding. Knockdown of ADAM10 in U87 CAR cells significantly decreased levels of constitutively shed CAR by 40% (Figure 5B). Similar results were obtained with the ADAM10 shRNA sequence #6675 (data not shown). PMA-mediated shedding of CAR also significantly decreased (by 41%) with knockdown of ADAM10 (Figure 5C), although it should be noted that the concentration and length of time used for the PMA treatments is considered to be chronic with a wide range of pleiotropic cellular effects; thus at these conditions, PMA activation of ADAM10 may be non-specific. Ionomycin-induced shedding of CAR ECD (following treatment of U87 CAR cells at 1.5 µM for 30 minutes) was also found to be ADAM10-dependent (Figure 5D). These data confirm that ADAM10 mediates constitutive, ionomycin-stimulated, and chronic PMA-stimulated 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)

shRNA knockdown of ADAM10 decreases constitutive, PMA-mediated and ionomycin-mediated CAR ECD shedding.(A) A Western blot for ADAM10 using cell lysates of U87 CAR cells containing either control shRNA or ADAM10 (#6676) shRNA, in biological duplicates. Equal amounts of proteins were loaded on SDS-PAGE gel. Anti-GAPDH antibody was used as a loading control. Quantification of mean ADAM10 band intensities normalized over GAPDH revealed a decrease of approximately 60% with anti-ADAM10 shRNA compared to control shRNA. (B) A constitutive shedding experiment was performed using U87 CAR stable cell lines containing either control shRNA or anti-ADAM10 shRNA (#6676). Conditioned media and cell lysates were collected after 24 hours of incubation of cells in opti-MEM, and Western blotting was done using the anti-CAR N-terminus antibody 2239. With anti-ADAM10 shRNA, there was a significant decrease (40%) in the levels of shed CAR. Results from 4 independent experiments performed in duplicates were quantified (unpaired t-test; p=0.0004 (***)). (C) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6676) were treated with 1 µM PMA. Conditioned media and cell lysates were collected after 3 hours, and Western blots were performed using the anti-CAR N-terminus antibody 2239. With shRNA knockdown of ADAM10, there was a significant decrease of 41% in levels of shed CAR compared to control shRNA. Results from 3 independent experiments performed in duplicates were quantified (one-way ANOVA with Tukey’s multiple comparison test; *** = p < 0.001). (D) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6675) were treated with 1.5 µM ionomycin (vs. DMSO vehicle). Conditioned media and cell lysates were collected after 30 minutes of treatment, and Western blots were performed using the anti-CAR N-terminus antibody 2240. With shRNA knockdown of ADAM10, there was a significant decrease of 52% in levels of shed CAR with ionomycin treatment compared to control shRNA. Results from 3 independent experiments (n=3 per group) were quantified (one-way ANOVA with Bonferroni’s multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).
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pone-0073296-g005: shRNA knockdown of ADAM10 decreases constitutive, PMA-mediated and ionomycin-mediated CAR ECD shedding.(A) A Western blot for ADAM10 using cell lysates of U87 CAR cells containing either control shRNA or ADAM10 (#6676) shRNA, in biological duplicates. Equal amounts of proteins were loaded on SDS-PAGE gel. Anti-GAPDH antibody was used as a loading control. Quantification of mean ADAM10 band intensities normalized over GAPDH revealed a decrease of approximately 60% with anti-ADAM10 shRNA compared to control shRNA. (B) A constitutive shedding experiment was performed using U87 CAR stable cell lines containing either control shRNA or anti-ADAM10 shRNA (#6676). Conditioned media and cell lysates were collected after 24 hours of incubation of cells in opti-MEM, and Western blotting was done using the anti-CAR N-terminus antibody 2239. With anti-ADAM10 shRNA, there was a significant decrease (40%) in the levels of shed CAR. Results from 4 independent experiments performed in duplicates were quantified (unpaired t-test; p=0.0004 (***)). (C) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6676) were treated with 1 µM PMA. Conditioned media and cell lysates were collected after 3 hours, and Western blots were performed using the anti-CAR N-terminus antibody 2239. With shRNA knockdown of ADAM10, there was a significant decrease of 41% in levels of shed CAR compared to control shRNA. Results from 3 independent experiments performed in duplicates were quantified (one-way ANOVA with Tukey’s multiple comparison test; *** = p < 0.001). (D) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6675) were treated with 1.5 µM ionomycin (vs. DMSO vehicle). Conditioned media and cell lysates were collected after 30 minutes of treatment, and Western blots were performed using the anti-CAR N-terminus antibody 2240. With shRNA knockdown of ADAM10, there was a significant decrease of 52% in levels of shed CAR with ionomycin treatment compared to control shRNA. Results from 3 independent experiments (n=3 per group) were quantified (one-way ANOVA with Bonferroni’s multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Mentions: To further confirm ADAM10’s role in CAR shedding, we used shRNA to knock down ADAM10 expression in U87 CAR cells. Stable cell lines were generated for anti-eGFP shRNA (used as control) or anti-ADAM10 shRNA (sequences #6675 or #6676). The shRNA sequences targeting ADAM10 successfully knocked down ADAM10 mRNA levels (Figure S3) and, subsequently, ADAM10 protein levels (Figure 5A). The shRNA stable cell lines were then used to study constitutive and regulated CAR ECD shedding. Knockdown of ADAM10 in U87 CAR cells significantly decreased levels of constitutively shed CAR by 40% (Figure 5B). Similar results were obtained with the ADAM10 shRNA sequence #6675 (data not shown). PMA-mediated shedding of CAR also significantly decreased (by 41%) with knockdown of ADAM10 (Figure 5C), although it should be noted that the concentration and length of time used for the PMA treatments is considered to be chronic with a wide range of pleiotropic cellular effects; thus at these conditions, PMA activation of ADAM10 may be non-specific. Ionomycin-induced shedding of CAR ECD (following treatment of U87 CAR cells at 1.5 µM for 30 minutes) was also found to be ADAM10-dependent (Figure 5D). These data confirm that ADAM10 mediates constitutive, ionomycin-stimulated, and chronic PMA-stimulated 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