Barnea et al., 1994;Maurel et al., 1994). It has been expressed on the surface of glial cells binds to the shown that the expression of RPTPβ is restricted to the glycosylphosphatidylinositol (GPI)-anchored recogninervous system. RPTPβ is expressed in cells that have been tion molecule contactin on neuronal cells leading to implicated in neuronal migration and axonal guidance, neurite outgrowth. We describe the cloning of a novel including glial precursors, radial glia and astrocytes (Rauch contactin-associated transmembrane receptor (p190 Canoll et al., 1993). RPTPβ also bears the Caspr) containing a mosaic of domains implicated in HNK-1 carbohydrate epitope that is found in several protein-protein interactions. The extracellular domain neuronal adhesion molecules and was implicated in cell of Caspr contains a neurophilin/coagulation factor recognition and axonal guidance (Rauch et al., 1991). In homology domain, a region related to fibrinogen β/γ, Drosophila, the analogous HRP carbohydrate epitope was epidermal growth factor-like repeats, neurexin motifs found in neural recognition molecules as well as in as well as unique PGY repeats found in a molluscan receptor protein tyrosine phosphatases that are expressed adhesive protein. The cytoplasmic domain of Caspr in the developing nervous system (Desai et al., 1994). It contains a proline-rich sequence capable of binding to was demonstrated recently that loss-of-function mutations a subclass of SH3 domains of signaling molecules.in Drosophila RPTPs result in erroneous pathfinding Caspr and contactin exist as a complex in rat brain of certain motor axons (Desai et al., 1996; Krueger and are bound to each other by means of lateral (cis) et al., 1996). interactions in the plasma membrane. We proposeIn our attempts to identify specific ligands of RPTPβ, that Caspr may function as a signaling component we used soluble, recombinant CAH or FNIII domains of of contactin, enabling recruitment and activation of this receptor phosphatase as specific reagents for the intracellular signaling pathways in neurons. The bindidentification of cellular proteins that bind to RPTPβ. We ing of RPTPβ to the contactin-Caspr complex could have demonstrated that the FNIII repeat binds specifically provide a mechanism for cell-cell communication to glial cells while the CAH domain of RPTPβ binds to between glial cells and neurons during development.neurons or cells of neuronal origin .

Specialized paranodal junctions form between the axon and the closely apposed paranodal loops of myelinating glia. They are interposed between sodium channels at the nodes of Ranvier and potassium channels in the juxtaparanodal regions; their precise function and molecular composition have been elusive. We previously reported that Caspr (contactin-associated protein) is a major axonal constituent of these junctions (Einheber et al., 1997). We now report that contactin colocalizes and forms a cis complex with Caspr in the paranodes and juxtamesaxon. These proteins coextract and coprecipitate from neurons, myelinating cultures, and myelin preparations enriched in junctional markers; they fractionate on sucrose gradients as a high-molecular-weight complex, suggesting that other proteins may also be associated with this complex. Neurons express two contactin isoforms that differ in their extent of glycosylation: a lower-molecular-weight phosphatidylinositol phospholipase C (PI-PLC)-resistant form is associated specifically with Caspr in the paranodes, whereas a higher-molecular-weight form of contactin, not associated with Caspr, is present in central nodes of Ranvier. These results suggest that the targeting of contactin to different axonal domains may be determined, in part, via its association with Caspr. Treatment of myelinating cocultures of Schwann cells and neurons with RPTP␤-Fc, a soluble construct containing the carbonic anhydrase domain of the receptor protein tyrosine phosphatase ␤ (RPTP␤), a potential glial receptor for contactin, blocks the localization of the Caspr/contactin complex to the paranodes. These results strongly suggest that a preformed complex of Caspr and contactin is targeted to the paranodal junctions via extracellular interactions with myelinating glia.

Receptor protein tyrosine phosphatase β (RPTPβ) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (βC) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, βCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than βCF. βS had no effect by itself, but it potentiated neurite growth when mixed with βCF. Neurite outgrowth induced by βCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound βCFS. NIH3T3 cells transfected to express βCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPβ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.