Ephrin-A5 is competent to transmit a compartmentalized signal event on binding to its cognate receptor. (A) NIH-3T3 cells expressing ephrin-A5 were incubated for the times indicated with either Fc or EphA5–Fc. Cells were lysed and fractionated on a sucrose gradient. Both caveolae-like fractions (C) and soluble fractions (S) were analyzed by Western blot with the 4G10 antibody. The blot was reprobed with an anti-caveolin-2 antibody to control for the amount of TX-100 insoluble material loaded. (B) Caveolae-like fractions purified from NIH-3T3 cells expressing ephrin-A5 that were stimulated with EphA5–Fc for the times indicated (minutes) were analyzed by Western blotting with the 4G10 antibody. The blot was reprobed with anti-caveolin-2 antibody to control for the amount of material loaded. Molecular mass markers are indicated in kD.

p80 is a putative substrate for Src-family kinases. (A) Caveolae-like fractions isolated from EphA5–Fc-stimulated fibroblasts were solubilized in TX-100 at 37°C for 30 min and incubated with 50 μg of either purified GST or GST–Src–SH2 fusion proteins. Protein complexes were pelleted by low-speed centrifugation and analyzed by Western blotting using the phosphotyrosine-specific antibody 4G10. (B) Caveolar fractions were isolated from ephrin-A5-expressing cells that had been treated with either Fc or EphA5–Fc in the presence or absence of PP1 (as indicated). Samples were analyzed as above with GST–Src–SH2 fusion protein and Western blotting with 4G10. Molecular mass markers are indicated (kD).

Binding of EphA5–Fc to ephrin-A5-expressing cells induces changes in cell architecture, requiring the Src-family kinases. (A) NIH-3T3 cells expressing ephrin-A5 (A5-2) (a,b) and primary murine astrocytes (c,d) were incubated with Fc (a,c) or EphA5–Fc (b,d) proteins for 30 min at 37°C, fixed and subjected to indirect immunofluorescence using anti-vinculin monoclonal antibody (clone VIN-11-5, Sigma) and a Cy³-conjugated anti-mouse secondary antibody. (B) NIH-3T3 cells expressing ephrin-A5 (A5-2) (a–f) were incubated with Fc (a,c,e) or EphA5–Fc (b,d,f) proteins for 30 min at 37°C, in the presence of either vehicle (DMSO) (a,b), 1 μm Gö 6983 (c,d), or 10 μm PP1 (e,f). The cells were then fixed with methanol and subjected to indirect immunofluorescence using anti-vinculin monoclonal antibody.

Model for the activation of ephrin-A ligands regulating cellular architecture. Ephrin-A5 is localized to discrete membrane microdomains. Upon binding to its cognate Eph receptor expressed on a neighboring cell, a signal is initiated within the ephrin-A5-expressing cell. This signal, presumably requiring the Rho family of small molecular weight GTPases, results in a change in cellular adhesion that may affect neuronal migration and guidance. The unknown p80 phosphoprotein, the Src-family kinase Fyn, and a putative transmembrane adapter protein (X) are indicated and presumably perform some essential functions during intracellular signaling.

Ephrin-A5 is compartmentalized within discrete plasma membrane microdomains. (A) NIH-3T3 cells stably expressing human ephrin-A5 were subjected to immunofluorescence using the Fc fragment of human IgG as negative control (a), the EphA5–Fc fusion protein (8 μg/ml) to detect the ephrin-A5 protein (b), or caveolin-1-specific monoclonal antibody (c). (B) NIH-3T3 cells ectopically expressing ephrin-A5 (A5-1) or wild type (WT) were lysed in 1% Triton X-100, and the caveolar (C) and Triton X-100 soluble (S) fractions were collected as described previously (Robbins et al. 1995) (left). The isolated caveolae-like fraction from ephrin-A5-expressing cells was incubated with 5 μg of either EphA5–Fc or Fc fusion proteins, pelleted by the addition of protein A–Sepharose and analyzed by immunoblotting (right). Both blots were probed with a monoclonal antibody to ephrin-A5 (clone 5G2). (C) The ephrin-A5-expressing fibroblasts were lysed in 1% Triton X-100 and fractionated by linear sucrose gradients (5%–40%) as above, except individual 1-ml aliquots were collected and subjected to a Western blot analysis using caveolin-1, caveolin-2, and ephrin-A5-specific antibodies as indicated. Molecular mass standards are labeled (kD).

Fyn tyrosine kinase is recruited to the caveolae-like domains upon ephrin-A5 engagement. (A) NIH-3T3 cells expressing ephrin-A5 were incubated for different times with EphA5–Fc, in the absence or presence of PP1 as indicated. Caveolae-like fractions (cav) were isolated and analyzed by Western blot using in parallel 4G10 and an antibody specific for Fyn. (B) NIH-3T3 cells expressing ephrin-A5 were fractionated on a sucrose gradient. Caveolae-like fractions (C) and soluble fractions (S) were tested by Western blot for the presence of Fyn and Src proteins. Molecular weight mass are indicated in kD.

Fyn tyrosine kinase is essential for signaling in caveolae-like microdomains in vivo. (A) Caveolae-like microdomains (C) and detergent soluble fractions (S) were isolated from cortex of mice at different stages of development (E18 and P3). Samples were analyzed by Western blot using 4G10, caveolin-2, and Fyn-specific antibodies. (B) Caveolae-like fractions (cav) were isolated from brain of mice that were genotyped wild type (Fyn^+/+), defective for Fyn kinase (Fyn^−/−), or heterozygous (Fyn^+/−). Fractions were analyzed by Western blotting using 4G10 and an antibody specific for caveolin-2.

Activation of ephrin-A5 results in an increase in cellular adhesion. Cells expressing ephrin-A5 (A5–2 and NG108–15) or cells transfected with the vector control (WT) were detached from their substratum by nonenzymatic removal, resuspended in low-serum containing media and spun down on plates coated with fibronectin. Cells were immediately incubated with either Fc (shaded bars) or EphA–Fc (solid bars) as indicated. Nonadhering cells were removed and the remaining adhering cells were trypsinized and counted. Values are expressed as the number of adhering cells from three different experiments (in triplicate).