Functional analysis of the MAP2 repeat domain.
The neuronal microtubule-associated protein MAP2 binds to microtubules via a domain near its C terminus containing a set of 3 or 4 imperfect repeats of a 31 amino acid motif. Using naturally occurring and mutated forms of the molecule containing between 1 and 4 repeats we have examined the contribution that these repeats make to MAP2 function and explored the significance of their repetition. The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are expressed in developing neurons and in glia respectively, and mutant 1- and 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the molecule unaltered. The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected non-neuronal cells and their ability to promote microtubule assembly in vitro. We found that each of the known effects of MAP2, including the bundling of microtubules and induction of process formation in living cells, are expressed by the 1-repeat form MAP2c3, which contains only the third repeat (R3). A second 1-repeat form, MAP2c4, which contains only R4, interacts more weakly with tubulin in vitro and does not bind to microtubules in transfected cells. The microtubule-related properties of MAP2 thus arise mainly from a single predominant repeat unit, R3. In vitro assembly experiments showed that the primary effect of all the repeats is to lower the critical concentration of tubulin required for microtubule assembly but that they differ greatly in potency. The results did not reveal a separate function related to the repetition of the repeat motifs, but instead suggest that its purpose is to tailor the efficiency of MAP2 to the cellular environment in which it has to function.
Ludin
B
B
Friedrich Miescher Institute, Basel, Switzerland.
Ashbridge
K
K
Fünfschilling
U
U
Matus
A
A
D000595:Amino Acid Sequence; D015870:Gene Expression / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014407:Tumor Cells, Cultured
D008869:Microtubule-Associated Proteins