Molecular Brain Research 77 (2000) 19–28 www.elsevier.com / locate / bres Research report The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain a,b,1 a,b,1 a a Takao Honda , Hiroshi Saitoh , Masayoshi Masuko , Takako Katagiri-Abe , a,b a c c Kei Tominaga , Ikuo Kozakai , Kazuo Kobayashi , Toshiro Kumanishi , b b a, * Yuichi G. Watanabe , Shoji Odani , Ryozo Kuwano a Research Laboratory for Molecular Genetics, Niigata University,1- Asahimachi, Niigata 951-8510, Japan b Course of Biosystem Science, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan c Brain Research Institute, Niigata University, Niigata 951-8585, Japan Accepted 25 January 2000 Abstract In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10.5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.  2000 Elsevier Science B.V. All rights reserved. Themes: Cellular and molecular biology Topics: Membrane composition and cell-surface macromolecules Keywords: Coxsackievirus and adenovirus receptor; Nerve growth cone; Adhesion molecule; Subcellular localization; In situ hybridization 1. Introduction system. Membrane proteins on nerve growth cones and growing axons act as recognition molecules involved in The transient expression of an adhesion molecule is a neurite extension, pathfinding and targeting of appropriate crucial event for neural network formation in the early cells, and consequently the formation of neural connect- developmental stages of the brain when neurons are most ions [7,11]. actively engaged in cell–cell interactions. Cell surface We purified membrane proteins exhibiting developmen- adhesion molecules are thought to play a crucial role in tal changes, and determined the partial amino acid se- axon guidance and fasciculation in the developing nervous quences of more than 130 peptides obtained on proteolytic cleavage of the membrane proteins [1]. Among them we focused on a membrane protein with a molecular mass of *Corresponding author. Tel.: 181-25-227-2343; fax: 181-25-227- 46 kDa, which was found to be expressed extensively in 0793. prenatal brains but to be decreased in adult brains on E-mail address: ryosun@gene.med.niigata-u.ac.jp (R. Kuwano) 1 These authors contributed equally to this work. Western blot analysis with antibodies against the purified 0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X(00)00036-X