Sema5A binds to plexin-B3 with high affinity and specificity. (A) Binding of the indicated recombinant semaphorins fused to AP (Sema–AP) to COS cells expressing either plexin-B1 (a,b), plexin-B2 (c,d) or plexin-B3 (e–h). Cell-bound AP activity was revealed in situ using NBT–BCIP substrates, as described in Methods. (B) Scatchard analysis of Sema5A binding to cells expressing plexin-B3. COS cells expressing plexin-B3 or vector alone were incubated with increasing concentrations of Sema5A–AP for 1 h. Cell-bound semaphorin was then measured as AP activity on the chromogenic substrate pNPP, as described in Methods. The results are the mean of triplicate data points, and are representative of three independent experiments. (C) The binding of the indicated proteins (fused to AP) to cells expressing plexin-B1, plexin-B2 and plexin-B3 was quantified using the chromogenic substrate pNPP. Results are representative of three experiments performed in duplicate with consistent results. (D) Plexin-B3-expressing cells were incubated with Sema5A–AP alone (10 nM) or in the presence of equimolar amounts of soluble TSP-1 domain, and the binding was quantified as in (C). TSP-1 repeats markedly reduced the binding of Sema5A to plexin-B3, whereas they did not interfere with the interaction between Sema4D and its receptor plexin-B1.

Sema5A triggers the collapsing response in fibroblasts expressing plexin-B3. (A) NIH-3T3 fibroblasts expressing plexin-B3 were incubated with 30 nM of oligomerized Sema5A–Fc or Sema7A–Fc (see Methods). Plexin-B1-expressing cells were treated in the same conditions with oligomerized Sema5A–Fc or with Sema4D. Cells were stained with crystal violet and examined using a Leica DMIL microscope. (B) The collapsing response shown in (A) was quantified by photographing at least four representative × 20 microscope fields from two independent experiments and counting the number of cells that underwent contraction (defined as rounded cells having a diameter equal to or less than 20 μm; see Barberis et al, 2004). The results are the means of cell counts per field.

Sema5A triggers Met signalling by interaction with plexin-B3. (A) Western blotting of immunopurified endogenous Met from parental MLP-29 cells and cells engineered to express plexin-B3. Sema5A triggers Met activation and autophosphorylation in a plexin-B3-dependent manner. This is also indicated by a slight band shift of phosphorylated proteins, as expected. (B) MLP-29 cell migration assay using Transwell^® inserts (see Methods). The indicated cell types were allowed to migrate towards oligomerized 10 nM Sema5A or 1 nM HGF in the presence of 5% FBS, or towards 20% FBS alone. After 16 h, migrated cells were fixed and stained with crystal violet. Cell migration was quantified by reading dye absorbance at 595 nm, and values were normalized to migration in control (ctr) medium (=100). Results are representative of three experiments performed in duplicate with consistent results. (C) Immunoblot analysis to demonstrate the expression of plexin-B3 (VSV-tagged) and Met in cells used for the experiments shown in (A,B). Met expression is barely detectable in cells subjected to targeted siRNA technology. (D) The chemotactic migration of primary HUVECs, either mock or infected to express plexin-B3, in response to either 4 nM HGF or 10 nM Sema5A added to the lower chamber of Transwell^® insert. Cell migration was quantified as in (B). The data are the means of two independent experiments.