Functional study of the cytoplasmic domain of coxsackie and adenovirus receptor (CAR)

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Coxsackie and adenovirus receptor (CAR), a binding receptor shared by subfamilies of Coxsackie viruses and adenoviruses, is an adhesion molecule which plays critical roles in development. CAR is also a putative tumor suppressor, and its expression is silenced or downregulated in a variety of tumor types. However, it is not known how CAR exerts its physiological functions. Our lab has previously reported that CAR is a potent suppressor of glioma cell migration, invasion and tumor growth in vivo, and that the cytoplasmic domain of CAR is required for these inhibitory effects, but the underlying mechanism is not entirely understood.In this thesis, we focused on characterizing the role of the four tyrosine residues, Y269, Y294, Y313 and Y318 in the cytoplasmic domain of CAR. These tyrosines were individually mutated to alanines, followed by expression in a CAR-negative glioma cell model. We found that while Y269 plays a role in mediating adenovirus infection, Y294 plays a role in STAT5 signaling during glioma cell migration. In the context of our studies on Y294, we observed that wild-type CAR inhibits STAT5 activation, its nuclear translocation, expression of its target genes and cell migration induced by growth factors, such as epidermal growth factor (EGF) and insulin growth factor 1(IGF-1). In contrast, Y294A abrogated the inhibitory function of CAR in STAT5 activation, target gene expression and cell migration, suggesting that Y294 is critical for CAR regulation of STAT5 signaling during cell migration. During these studies we also observed that introduction of wild-type CAR into various glioma cell lines invariably resulted in a decrease in the size of the cell nucleus. Three-dimensional reconstruction of confocal images further revealed that nuclear volume was diminished in CAR-expressing cells. Importantly, specific knockdown and conditional knockout of CAR in primary astrocytes resulted in increased nuclear size, suggesting that CAR-mediated nuclear size control is physiologically relevant. CAR localization on the plasma membrane was required for its effect on nuclear size, which could be abrogated by incubation of cells with a neutralizing antibody against the extracellular domain of CAR. We show that an intact cytoplasmic domain, specifically the distal 26 amino acids, is essential for control of the nuclear size in CAR-expressing cells. This domain was previously shown by our lab to interact with actin. Here we provide biochemical and electron microscopic evidence that CAR controls nuclear size and shape through its impact on the polymerization and bundling of the perinuclear actin network. While other tyrosine mutants have the same effect as CAR in reducing nuclear size, the CAR-Y318A does not reduce nuclear size, suggesting that Y318 might play a role in regulating CAR-mediated nuclear size control.In summary, we found that the tyrosine residues in the cytoplasmic domain of CAR play critical roles in various physiological processes, including cell migration, virus infection and nuclear size control. This study extends our understanding on the roles of CAR in cell migration and nuclear size control.

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