Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched clathrin assembly polypeptide.

NP185, a neuronal-specific protein of 185 kDa, was first discovered when we prepared monoclonal anti-bodies (mAbs) against bovine brain clathrin coated vesicles. Two mAbs, 8G8 and 6G7, permitted us to characterize this protein both biochemically and in development (NP185 is expressed in a NGF-dependent manner in PC12 cells). The expression of NP185 coincides with synaptogenesis. In this work, we have further characterized this protein as follows: Microsequence analysis of immuno-purified native NP185 from bovine brain yielded five peptides that corresponded exactly to the known sequences of murine F1-20 and rat AP180 (renamed AP3); ii) Using an established assay, we show that purified recombinant NP185/AP3 can facilitate clathrin cages assembly; iii) Using deletion mutagenesis, we mapped the epitopes of two distinct mAbs directed against bovine NP185 to a 60 amino acid residue region of the murine recombinant NP185/AP3; iv) Recombinant NP185/AP3 can be phosphorylated by purified casein kinase II in vitro; and v) Recombinant NP185/AP3 directly binds to purified brain tubulin. Since NP185/AP3 binds to tubulin and stimulates the clathrin assembly, it may be involved in the regulation of the transport of clathrin-coated vesicles. Casein kinase II, an enzyme known to be present in clathrin-coated vesicles, may play a role in the regulation of NP185/AP3 for the promotion of clathrin assembly.