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 mutat ed forms of the molecule containing between 1 and 4 repeats we have ex amined the contribution that these repeats make to MAP2 function and e xplored the significance of their repetition, The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are ex pressed in developing neurons and in glia respectively, and mutant 1-a nd 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the mo lecule unaltered, The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected no nneuronal cells and their ability to promote microtubule assembly in v itro, We found that each of the known effects of MAP2, including the b undling of microtubules and induction of process formation in living c ells, are expressed by the 1-repeat form MAP2c3, which contains only t he third repeat (R3), A second 1-repeat form, MAP2c(4), which contains only R4, interacts more weakly with tubulin in vitro and does not bin d to microtubules in transfected cells, The microtubule-related proper ties of MAP2 thus arise mainly from a single predominant repeat unit, R3, In vitro assembly experiments showed that the primary effect of al l the repeats is to lower the critical concentration of tubulin requir ed for microtubule assembly but that they differ greatly in potency. T he results did not reveal a separate function related to the repetitio n of the repeat motifs, but instead suggest that its purpose is to tai lor the efficiency of MAP2 to the cellular environment in which it has to function.