MYELIN PROTEIN ZERO--MECHANISMS OF MEMBRANE ADHESION

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Protein zero (P-0, mw = 28,500) is the major adhesive glycoprotein of the myelin sheath in the mammalian peripheral nervous system. Mutations in the P0 gene in humans underlie certain myelin diseases that affect the peripheral nervous system (Charcot-Marie-Tooth and Dejerine-Sottas Diseases). P0 is also the "simplest" transmembrane member of the immunoglobulin gene (Ig) superfamily, and so this molecule has attracted great interest as a prototypic representative of Ig superfamily interactions. Definitively establishing the structural basis for P0:P0 membrane adhesive interactions will have direct bearing on understanding the binding elements by which other Ig superfamily molecules exert their' actions. Our single Specific Aim is to experimentally evaluate a proposed model for P0:P0 molecular interactions which lead to strong membrane adhesion. In the first phase of these studies, we will engineer full length P0 cDNAs encoding polypeptides mutagenized by point mutation in the extracellular domain. Strategic mutations will be placed such that we may test the roles for amino acid residues at several specific contact points first revealed by analysis of the P0 extracellular domain. Each mutagenized cDNA will be used as template for mRNA synthesis, which in turn will program a coupled frog oocyte system we devised to test for intermembrane adhesion. In parallel studies, permanent expressing pure clonal cell lines of mutagenized P0 expressors will be used in cellular assays for adhesion and aggregation in normally non-adherent, non-aggregating cells such as HeLa or L-cells.