Induction of L1-CAM-mediated EGFR tyrosine kinase activity in S2 cells. Protein expression in transfected S2 cell lines was induced overnight in serum-free medium. Cells were briefly aggregated and prepared for immunocytochemistry with antiphosphotyrosine antibodies. (A-D) Aggregates of S2 cells that coexpress human L1-CAM and human EGFR; (E-H) S2 cells that express human L1-CAM; (I-M) S2 cells that coexpress Drosophila Fasciclin I and human EGFR. Bar, 12 μm.

Model of the functional interactions between L1-type CAMs and EGF receptor proteins. L1-cell adhesion relies on trans-interactions between L1-type proteins, which are expressed on the surface of neighboring cells. The interaction between L1-type CAMs and neuronal EGFR involves cis-, as well as trans-binding events. Cis-interactions between the two molecules are required for the activation of EGFR tyrosine kinase activation. A current model of L1-CAM homophilic cell adhesion also involves both cis- and trans-interactions (Kamiguchi and Lemmon, 2000).

S2 cells expressing human EGFR interact with S2 cell aggregates, which express L1-CAM. Shown are representative results of an S2 cell mixing experiment, in which unlabeled S2 cells that either expressed human L1-CAM (A-C) or Drosophila Fasciclin I (D) were coaggregated with DiI-labeled S2 cells, which either expressed human L1-CAM (A), no exogenous S2 cell protein (B), or human EGFR (C and D). Cell aggregates of 10 and more cells were counted as positive when they contained 3 or more DiI-labeled S2 cells; e.g., the larger cell aggregate in C was scored as positive, whereas the smaller aggregate as negative. Bar, 70 μm.

Human EGFR which is expressed by Drosophila S2 cells can be activated by the addition of 100 ng/ml human EGF to the tissue culture medium. Native or transfected S2 cell lines were induced overnight with CuSO₄ and recombinant human EGF (Sigma, St. Louis, MO) was added for 30 min to nonaggregated cells at a final concentration of 100 ng/ml. Subsequently, the cells were harvested for SDS-polyacrylamide electrophoresis and Western blot analysis with an antiphosphotyrosine antibody. Each lane contains the total protein from 2.5 × 10⁵ cells.

The heterophilic trans-interaction between human EGFR and human L1-CAM does not induce EGFR tyrosine kinase activity. Shown are three series of overlapping micrographs. Each first image was taken using Nomarski optics. The second panels display the same area as shown by Nomarski optics using epifluorescence microscopy with a rhodamine filter set. This visualizes S2 cells, which express human EGFR. The third image of each series was obtained with an FITC filter set and represents phosphotyrosine staining. Set A shows a positive control of S2 cells, which coexpress both human EGFR and L1-CAM. Panel sets B and C are from a mixing experiment, in which S2 cells expressing human L1-CAM were coaggregated with S2 cells, which expressed only transfected human EGFR. Bar, 15 μm.

Western blot analysis of transfected Schneider 2 cell lines. Panel A demonstrates that S2 cells do not express Drosophila EGFR. Shown is a Western blot that was incubated with a rat anti-Drosophila DER antiserum. Lane 1: the membrane protein from 2.5 × 10⁵ S2 cells; lane 2: 30 μg of total Drosophila embryonic protein. Bound antibodies were visualized using the ECL kit from Amersham Pharmacia Biotech. Immunoblots B-D show that transfected S2 cells express human L1-CAM, human EGFR, and/or Drosophila Fasciclin I. Each lane contains the total protein from 2.5 × 10⁵ induced S2 cells. Lanes 1: native S2 cells; lanes 2: S2 cells expressing human EGFR; lanes 3: S2 cells expressing human L1-CAM; lanes 4: S2 cells expressing human EGFR and human L1-CAM; lanes 5: S2 cells expressing Drosophila Fasciclin I; and lane 6: S2 cells expressing human EGFR and Drosophila Fasciclin I. Western blot B was incubated with a goat anti-EGFR antiserum, Western blot C with a goat anti-L1-CAM antiserum, and Western blot D with the f5H7 MAb against Drosophila Fasciclin I.

Axonal growth and pathfinding phenotypes in the developing Drosophila wing. (A-H) Each wing has a different genotype, as indicated in each panel. The L1 nerve at the anterior margin of the wing and the L3 nerve along the third wing vein were immunochemically visualized using the MAb 22C10. The wings in A-D exhibit a wild-type nerve pattern and represent controls for the various transgenic lines used in this experiment. (E and F) Typical examples of the axonal growth and pathfinding alterations, which are caused by the overexpression of the Nrg¹⁶⁷ isoform (E) or of the artificial GPI- anchored isoform Nrg^GPI (F). Inappropriate axonal connections between the L1 and the L3 nerve are marked by arrowheads. Axonal nodules and axons that perforate the wing epithelium are indicated by arrows. (G-H) Wings from pupae with the hypomorphic DER mutation torpedo in either homo- or heterozygous configuration, respectively. Although these wings are still smaller than wild-type, they do not exhibit axonal growth or pathfinding alterations. Bar, 120 μm.

EGFR is phosphorylated at tyrosine residues in response to L1-CAM cell adhesion. (A) L1-mediated cell adhesion specifically induces the phosphorylation of human EGFR, which is expressed by Drosophila S2 cells. After protein induction, transfected S2 cells were either left nonaggregated at a low cell density in a tissue culture dish (lane 1) or were aggregated on a shaking platform for 20 min (lanes 2-5). Each lane represents the total protein extract from 3.5 × 10⁵ cells, which were separated by SDS-PAGE and tested for their phosphotyrosine content by Western blot analysis. Cells, which were analyzed in A, coexpressed human L1-CAM and human EGFR (lanes 1 and 2), Drosophila Fasciclin I and human EGFR (lane 3), or expressed only human L1-CAM (lane 4) or human EGFR (lane 5). (B) Human EGFR, which is immunoprecipitated from S2 cell aggregates expressing human L1-CAM, contains phosphorylated tyrosine residues. Shown is an immunoprecipitation experiment from S2 cell aggregates expressing either human L1-CAM (lanes 1 and 2) or Drosophila Fasciclin I (lanes 3 and 4) in conjunction with human EGFR. Proteins were solubilized with Triton X-100 and immunoprecipitated with either an anti-EGFR antiserum (lanes 1 and 3) or with a goat nonimmune serum (lanes 2 and 4). Both Western blots were probed with antiphosphotyrosine antibodies.