The neural cell adhesion molecule NCAM plays a critical role in mediating cell-cell interactions in the developing nervous system. Our laboratory has used monoclonal antibodies to characterize the structure-function relationship of NCAM, and to demonstrate for the first time that NCAM binds to heparan sulfate proteoglycan (HSPG). Monoclonal antibodies which recognize the heparan sulfate-binding domain of NCAM inhibit NCAM function, which provides strong evidence that HSPG participates in NCAM-mediated cell interactions during neural development. In the proposed study the neuronal HSPG(s) which interacts with NCAM will be identified and characterized. Conventional biochemical analysis of the HSPG will provide insight into the type of HSPG involved in cell-cell interactions in the developing nervous system. Neuronal HSPGs will be fractionated by virtue of the ability to co-purify with NCAM. Monoclonal and polyclonal antibodies will be produced which recognize the HSPG that binds to NCAM. This will permit the investigation of the role of HSPG in NCAM function using in vitro and in vivo functional assays. The antibodies will also be employed for he immunocytochemical analysis of the expression of the HSPG during neural development. The latter studies will provide information about the cellular distribution of a specific class of HSPG that is involved in NCAM-mediated cell adhesion.
A second aim of the proposal is to examine the mechanism by which HSPG participates in NCAM function. Previous studies have employed lipid vesicles containing purified NCAM, in liposome aggregation assays, to demonstrate that NCAM mediates cell adhesion by a homophilic binding mechanism. In the proposed study we will examine whether HSPG co-purifies with NCAM because of its ability to bind specifically to NCAM. Liposome aggregation assays will then be conducted using purified NCAM (treated to remove HSPG) and HSPG to determine whether HSPG introduction into NCAM-containing liposomes alters NCAM binding affinity, and hence the rate of cell-cell adhesion.
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| Burg, M A; Cole, G J (1994) Claustrin, an antiadhesive neural keratan sulfate proteoglycan, is structurally related to MAP1B. J Neurobiol 25:1-22 |
| Cole, G J; McCabe, C F (1991) Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth. Neuron 7:1007-18 |
| Reyes, A A; Akeson, R; Brezina, L et al. (1990) Structural requirements for neural cell adhesion molecule-heparin interaction. Cell Regul 1:567-76 |
