Characterization of the cellular receptor for coxsackievirus and adenovirus

Abstract: CAR, the cellular receptor for coxsackievirus and adenovirus, was discovered in 1997. CAR is an evolutionary conserved transmembrane protein that belongs to the CTX subfamily within the large immunoglobulin superfamily. At the intracellular tail, CAR has a PDZ domain binding motif suggesting that CAR is part of a larger protein complex. CAR is a cell adhesion molecule that binds to CAR on neighbouring cells. In polarized epithelial cells CAR is located at tight junctions where it recruits ZO-1, MUPP-1 and other scaffolding proteins. JAML, a CTX family member also interacts with CAR and this interaction is important in mediating neutrophil transmigration across tight junctions. During development CAR is highly expressed in several tissues. CAR is essential for heart development since CAR-deficient mice die early during embryonic development due to heart failure. In the adult, CAR is mainly confined to tight junctions present in epithelial cells. CAR may also function as a tumour repressor in certain cancer cell types. The aim of this thesis was to understand the biological role of CAR. In the first study in this thesis, three alternative splice variants of CAR were identified including a novel splice variant CAR-3 that appeared to be only expressed in the mouse heart. Also several conserved genomic regions within the human and mouse CAR gene were identified that might harbour transcriptional regulatory regions. In the second and third study a yeast two-hybrid screen was used to identify interacting partners towards the intracellular tail of CAR. Two proteins, LNX and LNX2 were identified and shown to interact with CAR both in vivo and in vitro. The LNX family interacts with Numb, a protein implicated in asymmetric cell division and Notch signalling. By deletion studies PDZ domain # 2 was identified both in LNX and LNX2 as important for binding to CAR. Deletion of the intracellular tail of CAR revealed that not only the PDZ binding motif, but also additional regions were involved in binding to the LNX proteins. Immunohistochemistry of a 16.5 day old embryo showed that both CAR and LNX2 were highly expressed during development and showed similar subcellular distribution in a subset of vessels. Finally, in the fourth study the role of CAR in the spermatogenesis process was investigated. CAR was detected in male germ cell in mice, rats and humans. The two major isoforms of CAR were seen in different sites in the acrosome of the spermatozoa suggesting that they might have different functions. CAR was detected on the surface of acrosome-reacted spermatozoa, suggesting that CAR may be involved in the fertilization process. LNX and LNX2 were also expressed in the spermatozoa. Finally CAR was found to form a complex with JAM-C, a CTXfamily member previously shown to be essential for polarization of round spermatids in the testis. In conclusion the results described in this thesis increases the knowledge of the structure and expression of the CAR gene and give clues to the biological function of CAR.