SEQUENCE-ANALYSIS OF MAP2 FUNCTION IN LIVING CELLS

Microtubule-associated protein 2 (MAP2) is an abundant neuron-specific protein that binds to microtubules through a domain near its carboxyl terminus that contains either three or four similar repeats of a 31 a mino acid motif. When expressed in non-neuronal cells by transfection MAP2 stabilises microtubules and induces their rearrangement into long bundles that are capable of supporting process outgrowth. To investig ate which elements in the MAP2 sequence are involved in these function s we have constructed a series of deletion mutants of the short embryo nic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased wi th the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which require d in addition contiguous sequences either amino-terminal or carboxyl-t erminal to the repeats themselves, Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise i ncreases in the length of neighbouring sequence produced a gradual inc rease in microtubule binding, The apparent strength of binding to micr otubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the re sulting microtubules to support process outgrowth, These results indic ate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the r epeat motifs and elements in the sequences on either side of them, who se additive effect produces the strong binding of the native MAP2 mole cule, The results further indicate that both the bundling and stiffeni ng of microtubules by MAP2 are correlated with the strength of its bin ding to them and suggest that these properties are a direct result of microtubule stabilisation.