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.
Highlights
The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule of the Immunoglobulin (Ig) superfamily [1,2]
CAR sheds its ectodomain To investigate the possibility that CAR ectodomain (ECD) is released from cells into the extracellular environment, we collected conditioned media from a human glioma cell line U87MG stably expressing CAR (U87 CAR), and performed Western blot analyses using rabbit polyclonal antibodies raised against the extracellular domain [11]
CAR was detected from lysates of U87 CAR cells (Figure 1A), while no CAR was detected from the control, vector-infected cell line (U87 LNCX)
Summary
The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule of the Immunoglobulin (Ig) superfamily [1,2]. CAR is highly conserved, especially in its C-terminus [2], and it is expressed in a variety of mammalian and non-mammalian species such as D. rerio and X. laevis. It is highly expressed in the developing nervous system, in neuronal growth cones [6,7], and it mediates neurite extension by binding to the extracellular matrix protein fibronectin [8]. CAR expression is critical for normal cardiac development, as its gene deletion in mice before embryonic day 11 results in cardiac abnormalities and embryonic lethality [13,14,15]. CAR deletion in adulthood leads to multiple organ phenotypes including impairment of cardiac atrioventricular connection [16,17], atrophy of exocrine pancreas, enlarged intestines, and an increase in the number of thymocytes in the thymus [18]
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