Sequence homology predicts that the extracellular domain of the sodium channel

Sequence homology predicts that the extracellular domain of the sodium channel β1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier. S2 cells induces aggregation which is proposed to occur through homophilic interactions between the extracellular domains of the β subunits (Malhotra et al. 2000 Our data demonstrate that β1 subunit extracellular domains can also associate heterophilically with another CAM. Determination of the neurofascin binding site The extracellular region of neurofascin 186 consists of multiple Olanzapine (LY170053) domains. To investigate which extracellular domains of neurofascin were able to interact with β1 the Ig domains Ig1-6 the FN domains FN1 2 and 4 and the mucin-like domain were expressed separately fused at the NH2 terminus to the HA.11 tag and at the COOH terminus to the GPI anchor sequence from human placental alkaline phosphatase. These constructs were coexpressed with sodium channel α and β1 subunits in tsA-201 cells. After immunoprecipitation with anti-SP20 only Ig1-GPI and FN2-GPI were able to associate with α/β1 complexes in tsA-201 cells suggesting that the neurofascin binding site is assembled from amino acids in both of these domains (Fig. 5 A). It was necessary to express β1 complexed with α subunits in these experiments as the GPI constructs were expressed at high levels and all of them bound nonspecifically to β1 in the absence of α subunits. When α subunits were expressed alone with Ig1-GPI or Olanzapine (LY170053) FN2-GPI no interaction was observed confirming that this association is β1 Olanzapine (LY170053) dependent (Fig. 5 C). Both the 155- and 186-kD isoforms of neurofascin contain Ig1 and FN2 so β1 should interact with both isoforms. However β1 is localized to nodes of Ranvier in sciatic nerve and is most likely to interact with neurofascin 186 which also concentrates at nodes. The interaction of Ig1 with β1 in cis suggests that the neurofascin molecule folds back on itself to make this domain accessible to β1 as drawn in Fig. 3 A; thus binding of amino acids on Ig1 and FN2 with β1 could then occur. It is also possible that the FN2 domain of neurofascin could interact with β1 subunits in a cis configuration whereas Ig1 interacts with β1 subunits at lower affinity in trans on an opposing membrane. Development of new methods to measure lower-affinity trans-interactions of β1 will be required to test this idea. Similar interactions are made by axonin 1/TAG-1-like glycoproteins a family of neural CAMs Rabbit Polyclonal to ACBD6. containing six Ig domains and four FN domains. Homophilic trans-interactions occur between Ig domains 2 and 3 of axonin 1 (Freigang et al. 2000 whereas the FN domains of TAX-1 the human homologue of axonin 1 are thought to form cis-homophilic interactions (Tsiotra et al. 1996 Figure 5. Determination of the neurofascin binding site. (A) TsA-201 cells were cotransfected with α and β1 subunits and GPI-tagged constructs Ig1-GPI Ig2-GPI Ig3-GPI Ig4-GPI Ig5-GPI Ig6-GPI FN1-GPI FN2-GPI FN4-GPI and mucin-GPI respectively. … These results demonstrate that the Olanzapine (LY170053) extracellular domain of β1 functions as a CAM by adhering to neurofascin. Neurofascin and NrCAM clusters appear along sciatic nerve axons at postnatal day 2 followed by recruitment of ankyrinG and sodium channels (Lambert et al. 1997 Sodium channel recruitment appears to be dependent on ankyrinG Olanzapine (LY170053) as sodium channels Olanzapine (LY170053) are no longer present at axon initial segments in the granule cells of ankyrinG-null mice and Purkinje cells show reduced ability to fire action potentials (Zhou et al. 1998 Therefore neurofascin and NrCAM may initially recruit ankyrinG and sodium channels may subsequently be targeted to these sites by the interactions between the extracellular domain of β1 and neurofascin and the intracellular domains of β1 and β2 with ankyrinG. We show that β1 subunits interact with neurofascin in developing rat brain and propose that this association is involved in targeting sodium channels to specialized regions of the neuron such as nodes of Ranvier and axon initial segments. Neurofascin and NrCAM are only able to associate with ankyrinG when the conserved tyrosine residue within the FIGQY sequence is dephosphorylated (Garver et al. 1997 We reported recently that sodium channels associate with receptor.