Common and distinct tubulin binding sites for microtubule-associated proteins.

Abstract

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.