Axons are insulated and are allowed to function efficiently by the multi-lamellar membrane, myelin. Peripheral nervous system myelin is held compact by P0, a protein that contain a single immunoglobulin (Ig)-domain. P0 holds myelin together at both the extracellular (via homophilic interactions) and at the cytoplasmic (putatively via interactions with lipids) membrane leaflets. Furthermore, by measuring the adhesion of P0 in transfected cells, a dynamic relationship between the interaction of the extracellular and cytoplasmic domain of P0 was demonstrated; the cytoplasmic domain must be intact, initially, interacting with the cytoskeleton, for adhesion of the extracellular sequences to take place. Similarly, P0 mutated in either the extracellular or the cytoplasmic domain of P0 can exert a dominant negative effect on adhesion, implying a cis interaction, within the membrane. Loss of any of these interactions of P0 could result in a loss of function of the molecule. Recently, mutations and deletions of P0 have been associated with the peripheral neuropathy. Charcot-Marie-Tooth (CMT),1B. The longterm objectives of this application are to characterize the interactions of P0 as a prototype for all Ig-members and to determine how mutations in P0 bring about CMT 1B. In this proposal, the cytoskeletal interactions will be investigated by identifying and characterizing P0-associated proteins, which may be a link to the cytoskleleton. Also, using a transfection/adhesion strategy, how post-translational modifications of P0 s cytoplasmic domain affect adhesion and cytoskeletal interactions will be addressed. The ability of P0 to cluster, an indication cis interactions, will be determined by cross-linking surface P0. In addition, monoclonal antibodies to a soluble, extracellular domain of P0, in its native form, will be raised and used to distinguish mutations that disrupt conformation from those that affect binding directly. Finally, with this information, how the mutations in P0 associated with CMT1B, affect its function will be addressed both in transfected cells and in transgenic mice. The various CMT1B-mutated P0 protein will be expressed in cells by transfection. Surface expression, clustering, antibody binding and adhesion will be assessed. Then the mutated proteins will be co-expressed with wild type P0 to determine if they exert a dominant negative effect. To complement these studies, transgenic mice, initially, expressing a tagged P0 will be examined both morphologically and biochemically. Eventually, mice co-expressing wildtype and mutant P0 will be produced and examined for a dysmyelination. In this way, how the various mutations in P0 bring about the disease phenotype in CMT1B can be addressed directly.
