Common and distinct tubulin binding sites for microtubule-associated proteins

A specific binding assay was developed that monitors the interaction of 125I-labeled microtubule-associated proteins (MAPs) with tubulin or its fragments bound to nitrocellulose membrane. To identify the tubulin-binding domains for MAPs we have examined the binding of rat brain 125I-labeled MAP2 or 125I-labeled tau factors to 60 peptides derived from porcine alpha- and beta-tubulin. MAP2 and tau factors specifically interacted with two peptides derived from the carboxyl-terminal region of beta-tubulin, which are located between positions 392-445 and 416-445. In addition, there is a distinct tau-binding site at the amino-terminal region of alpha-tubulin. tau factors but not MAP2 displayed strong interaction with a peptide derived from the amino-terminal domain of alpha-tubulin between positions 1 and 75. To narrow down the location of the beta-tubulin binding site that is common to MAP2 and tau factors, we have synthesized five peptides that are homologous to the corresponding sequence from the porcine or rat carboxyl-terminal region. Binding studies with the synthetic peptides suggest that amino acid residues 434-440 of beta-tubulin are crucial for the interaction of MAP2 and tau factors.