Figure 3.

AMAP1 associates with the β1 integrin subunit through PRKD2. (A) MDA-MB-231 cells plated onto collagen I–coated dishes were prestarved for serum for 2 h and then treated or untreated with 10 ng/ml EGF (5 min). Cells were lysed and anti-AMAP1 immunoprecipitates were analyzed for coprecipitation of PRKD2 and several integrin subunits (β1, β3, α2, α3, and α5) by immunoblotting, as indicated. Pre-immune serum of the anti-AMAP1 antibody was used as a control (Pre-imm). (B) Anti-AMAP1 immunoprecipitates from MDA-MB-231 cells overexpressing GST-C1 or GST-C4, prestarved and treated with EGF, were analyzed as above. Coprecipitation of PRKD2 and β1 integrin with AMAP1 was analyzed by immunoblotting using the indicated antibodies. In A and B, blots of whole cell lysates (WCL, 10 µg) are also shown. (C and D) Lysates from 293T cells expressing V5-PRKD2 or AMAP1-HA were mixed in different ratios, as indicated, and incubated in vitro with the GST-tagged cytoplasmic region of β1 integrin (GST-β1T) bound to glutathione beads. Precipitates were analyzed by immunoblotting using anti-V5, HA, and GST antibodies. GST alone was used as a control. (E–G) MDA-MB-231 cells expressing V5-PRKD2 were cultured on collagen I–coated dishes. Cells were serum-starved, and then treated (F) or untreated (E) with 10 ng/ml EGF (5 min) before fixation and immunolabeling. In the merged picture, V5-PRKD2, β1 integrin, and AMAP1 are shown as green, red, and blue, respectively. Bar, 10 μm. Colocalization of these proteins in serum-starved cells (open bars) and EGF-stimulated cells (closed bars) was measured, in which 1.0 and −1.0 indicate perfect colocalization and exclusion, respectively. Results represent mean ± SEM of >10 measurements (error bars).