NEUREGULINS AND AXONAL/GLIAL INTERACTIONS

New York University School Of Medicine, New York NY

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Abstract

The long term goal of this project is to identify and characterize the membrane components of axons and glial cells that mediate the cell-cell interactions of myelination in order to understand the normal development and the acquired disorders of myelinated nerve fibers. A major portion of our studies, has focused on the role of the myelin-associated glycoproteins (MAG). These proteins are integral membrane proteins of myelinating glial cells that are uniquely positioned to mediate the cell interactions of myelination - binding to specific axons via their ectodomain and, potentially, transducing intracellular signals through distinct cytoplasmic segments. To clarify MAG's physiologic role in myelination, we will characterize the structural elements that mediate and may regulate its adhesive function and identify the cytoplasmic constituents with which it interacts. In parallel studies, we have been characterizing surface components of those axons that are destined to become myelinated, with the view that proteins unique to these nerve fibers will provide important insights into their ensheathment fate by the glial cell and, in particular, the adhesion mediated by MAG. We have recently identified an apparently novel axonal protein that has a restricted fiber type distribution that we propose to characterize further. Specifically we will study wild type and recombinant MAG proteins (generated by site directed mutagenesis) in adhesion assays and in coprecipitation studies to: 1) identify the binding sequences of the ectodomain of MAG that promote adhesion, 2) determine whether this adhesion is regulated by the cytoplasmic segment, 3) determine if the two isoforms of MAG, that differ in their cytoplasmic sequences, are targeted to different membrane domains in cells, 4) determine whether these two isoforms differentially associate with cytoplasmic constituents, including kinases, 5) investigate the functional significance of the differential phosphorylation of the MAG isoforms and 6) investigate the axonal regulation of this phosphorylation. We will also purify and isolate cDNA clones for the novel PI linked protein of sensory nerve fibers that we have identified. Purified protein will be microsequenced and corresponding degenerate oligonucleotides will be synthesized and used to screen a cDNA library. cDNA clones will be isolated, sequenced, and used in functional assays. In parallel studies, the purified protein will be used in adhesion assays to determine its role in cell adhesion.