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 amino 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 investigate which elements in the MAP2 sequence are involved in these functions we have constructed a series of deletion mutants of the short embryonic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased with the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which required in addition contiguous sequences either amino-terminal or carboxyl-terminal to the repeats themselves. Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise increases in the length of neighbouring sequence produced a gradual increase in microtubule binding. The apparent strength of binding to microtubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the resulting microtubules to support process outgrowth. These results indicate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the repeat motifs and elements in the sequences on either side of them, whose additive effect produces the strong binding of the native MAP2 molecule. The results further indicate that both the bundling and stiffening of microtubules by MAP2 are correlated with the strength of its binding to them and suggest that these properties are a direct result of microtubule stabilisation.
Ferralli
J
J
Friedrich Miescher Institute, Basel, Switzerland.
Doll
T
T
Matus
A
A
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000502:physiology*; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure; D016296:Mutagenesis; D051381:Rats; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured
D008869:Microtubule-Associated Proteins