Integrin-alpha3 mediates binding of Chordin to the cell surface and promotes its endocytosis

Abstract

Dorsoventral patterning in animal development is regulated by a morphogenetic gradient of Bone morphogenetic protein signalling, which is established by a set of proteins that are conserved from Drosophila to vertebrates. These include Chordin (Chd)/Short gastrulation, Xolloid/Tolloid and Twisted gastrulation. Here, we report the identification of a cell-surface component of this morphogenetic pathway. Prompted by the observation that Chd protein bound to the surface of certain cell lines with subnanomolar affinity, we identified two cell-surface proteins that bind to Chd, one of which corresponds to Integrin-alpha3. Integrin-alpha3 and Chd are co-expressed in the Xenopus embryo. Transfection of Integrin-alpha3 increased the binding of Chd to the cell surface, which was competed by an excess of soluble Integrin-alpha3. After binding to the cell surface, Chd was translocated into intracellular endocytic compartments in a temperature-dependent manner. We propose that Integrin-alpha3 may regulate the concentration of Chd protein in the extracellular space by endocytosis.

Figure 1

Chordin binds to the cell surface. (A) Chordin–alkaline-phosphatase (Chd–AP) binds to COS-7 cells. COS-7 cells were incubated with Chd–AP or secreted AP (s-AP) for 2 h at 4 °C, and the amount of activity of bound AP was quantified using a fluorescent AP substrate. Equal loading was confirmed by protein measurements. (B) Binding curve and Scatchard analysis of Chd–AP binding to COS-7 cells. A dissociation constant (K_d) of 0.24 nM was estimated. (C) COS-7 cells were incubated with Chd–AP in the presence of 60 nM recombinant Integrin-α3 (Int-α3; Chemicon) or mouse Chd (R&D Systems), and the amount of bound Chd–AP was quantified using a fluorescent AP substrate. Other cysteine-rich-repeat-containing proteins were not tested because they are not available in purified form. (D) Chd binding requires an intact cell membrane. COS-7 cells were incubated with 0.1% or 1.0% Triton X-100 (TX-100), and Chd–AP was bound and quantified. (E) Chd binding colocalizes with actin filaments. 10T1/2 fibroblasts were incubated with pure Chd–Fc at 4 °C, fixed and permeabilized, and binding was visualized with an anti-human IgG antibody conjugated to Alexa Fluor 486. (F) The same cell as in (E), stained with phalloidin-Alexa558 to visualize actin. (G) Merge of (E) and (F). Note the overlap between Chd–Fc and actin (yellow).

Figure 2

Chordin affinity columns bind biotinylated cell-surface Integrin-α3. (A) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. (B) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. (C–G) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin (C,D) and integrin-a3 (E,F) expression at stages 10.5 and 32. (G) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.

Figure 3

Chordin binds to the cell surface through Integrin-α3. (A) Binding of Chordin–alkaline-phosphatase (Chd–AP) to P19, COS-7 and CHO cell lines. The inset shows an anti-Integrin-α3 immunoblot of cell extracts from the three cell lines. The amount of Chd–AP binding correlates with endogenous levels of Integrin-α3. (B,C) Chd–AP binds to 293T cells transfected with integrin-a3. 293T cells were transfected with LacZ or human integrin-a3 full-length complementary DNAs, incubated with recombinant Chd–AP and stained for AP activity. The inset shows an anti-Integrin-α3 (Int-α3) immunoblot of the corresponding cell extracts.

Figure 4

Chordin–Fc translocates into intracellular endocytic compartments at 37 °C. Cells were incubated on ice with Chordin (Chd)–Fc for 2 h, washed, and then incubated with Texas-Red–dextran for 30 min on ice (A–D) or at 37 °C (E–H). (A,E) Localization of Chd–Fc (green) bound to COS-7 cells. (B,F) Localization of Texas-Red–dextran (red) in punctate endocytic compartments after shifting to 37 °C. (C,G) Merged images. (D,H) 4,6-diamidino-2-phenylindole (DAPI) staining of nuclear DNA. Note that at 4 °C, most of the Chd–Fc is detected at the cell surface, but after 30 min at 37 °C it colocalizes with endocytic vesicles in the cytoplasm that contain Texas-Red–dextran (G). The red spot in (B) is artefactual, but shows that red fluorescence does not bleed into the green fluorescence channel.