STUDY OF THE INTERACTION OF THE MYELIN MONOMERIC CTP-BINDING PROTEINSWITH OTHER BRAIN PROTEINS

Although several monomeric GTP-binding proteins have been found in mye lin, the signaling pathways in which they operate are not known. To de fine these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-pol yacrylamide gel electrophoresis and blotted onto nitrocellulose membra nes, The presence of proteins that interact with the myelin GTP-bindin g proteins was explored by incubating those blots with an enriched fra ction of 22- and 25-kDa myelin GTP-binding proteins labeled with radio active guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa o ligodendrocyte polypeptides. Only the 28-kDa protein was present in my elin. In the active state (GTP-bound), they interacted only with a 47- kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kD a proteins in oligodendrocytes and total homogenate. Under these exper imental conditions the 28-kDa protein did not interact with the GTP-bi nding proteins. The fact that the myelin GTP-binding proteins in the a ctive state formed complexes with a different set of proteins than whe n in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptide s were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immu noblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an ef fector protein that is known to regulate the activation and movement o f several GTP-binding proteins between different cellular compartments . Thus, this study opens the way to identify the macromolecules partic ipating in the myelin signaling pathway involving monomeric GTP-bindin g proteins.