Heterotypic complex formation between subunits of microtubule-associated proteins 1A and 1B is due to interaction of conserved domains

AbstractThe microtubule-associated proteins MAP1A and MAP1B are related but distinct multi-subunit protein complexes that consist of heavy and light chains. The predominant forms of these complexes are homotypic, i.e. they consist of a MAP1A heavy chain associated with MAP1A light chains or a MAP1B heavy chain associated with MAP1B light chains, respectively. In addition, MAP1A and MAP1B can exchange subunits and form heterotypic complexes consisting of a MAP1A heavy chain associated with MAP1B light chains which might play a role in a transition period of neuronal differentiation. Here we extend previous findings by confirming that heterotypic MAP1B heavy chain-MAP1A light chain complexes also exist in the developing murine brain. We show that these complexes form through interaction of homologous domains conserved in heavy and light chains of MAP1A and MAP1B. Likewise, conserved domains of the MAP1A and MAP1B light chains account for formation of light chain heterodimers. By yeast 2-hybrid analysis we located the light chain binding domain on the heavy chain to amino acids 211–508, thereby defining a new functional subdomain

The light chains of microtubule-associated proteins MAP1A and MAP1B interact with α1-syntrophin in the central and peripheral nervous system.

Microtubule-associated proteins of the MAP1 family (MAP1A, MAP1B, and MAP1S) share, among other features, a highly conserved COOH-terminal domain approximately 125 amino acids in length. We conducted a yeast 2-hybrid screen to search for proteins interacting with this domain and identified α1-syntrophin, a member of a multigene family of adapter proteins involved in signal transduction. We further demonstrate that the interaction between the conserved COOH-terminal 125-amino acid domain (which is located in the light chains of MAP1A, MAP1B, and MAP1S) and α1-syntrophin is direct and occurs through the pleckstrin homology domain 2 (PH2) and the postsynaptic density protein 95/disk large/zonula occludens-1 protein homology domain (PDZ) of α1-syntrophin. We confirmed the interaction of MAP1B and α1-syntrophin by co-localization of the two proteins in transfected cells and by co-immunoprecipitation experiments from mouse brain. In addition, we show that MAP1B and α1-syntrophin partially co-localize in Schwann cells of the murine sciatic nerve during postnatal development and in the adult. However, intracellular localization of α1-syntrophin and other Schwann cell proteins such as ezrin and dystrophin-related protein 2 (DRP2) and the localization of the axonal node of Ranvier-associated protein Caspr1/paranodin were not affected in MAP1B null mice. Our findings add to a growing body of evidence that classical MAPs are likely to be involved in signal transduction not only by directly modulating microtubule function, but also through their interaction with signal transduction proteins

α1-syntrophin is found in a complex with LC1 in the central and peripheral nervous system.

<p>Brain protein extracts obtained from transgenic mice expressing myc-tagged LC1 were immunoprecipitated (<i>IP</i>) either with anti-myc antibodies (<i>anti-myc</i>) or without antibody (<i>no ab</i>; negative control). Pellets (<i>P</i>) and the corresponding supernatants (<i>S</i>) were fractionated by SDS-PAGE and analyzed by immunoblotting (<i>WB</i>) using anti-syntrophin (<i>anti-syn1351</i>) or anti-myc antibodies (<i>anti-myc</i>). The positions of protein size markers, syntrophin, and LC1 are indicated. The double band corresponding to LC1 resulted from insufficient denaturation prior to gel electrophoresis.</p

α1-syntrophin binds to microtubules in cells expressing LC1.

<p>PtK2 cells were transiently transfected to express either EGF-tagged α1-syntrophin alone (a and b) or α1-syntrophin and myc-tagged LC1 (c–e). Cells were fixed, co-stained for tubulin (<i>anti-tubulin</i>) and LC1 (<i>anti-myc</i>) and analyzed by fluorescence microscopy. In the absence of ectopically expressed LC1, α1-syntrophin was diffusely distributed throughout the cytoplasm (b). When co-expressed with LC1, α1-syntrophin was found to co-localize with LC1 on microtubules (c-e, arrows). Expression of LC1 causes microtubules to bundle, as has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049722#pone.0049722-Tgel1" target="_blank">[5]</a>. Scale bar, 20 µm.</p

MAP1B and syntrophin co-localize at the nodes of Ranvier and the abaxonal Schwann cell membrane.

<p>Sciatic nerves were prepared from 4-day (<i>4d</i>) or 14-day (<i>14d</i>) old or adult (<i>adult</i>) wild-type (<i>WT</i>) and MAP1B^−/− (<i>KO</i>) mice. Individual myelinated axons were isolated and stained for MAP1B (antibody anti-HC750) or syntrophin (pan syntrophin antibody anti-syn1351) as indicated. The pictures represent projections of confocal Z-stacks. The staining for MAP1B in postnatal and adult Schwann cells is specific as it is absent in Schwann cells of MAP1B^−/− mice. At all ages MAP1B was found to be concentrated at the nodes of Ranvier (<i>asterisks</i>). It also localized at the abaxonal membrane (<i>arrow heads</i>), particularly strong at postnatal day 14. Syntrophin was also found at nodes of Ranvier and the abaxonal membrane. In the adult, it was found to be localized to Cajal bands (<i>arrows</i>) in agreement with previous results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049722#pone.0049722-Albrecht1" target="_blank">[44]</a>. Co-localization of MAP1B and syntrophin was most prominent at the nodes of Ranvier and partial co-localization was found at the abaxonal membrane (<i>arrow heads</i>). Scale bar, 20 µm.</p