RCP does not affect integrin ligand-binding ability but mediates an association between α5β1 and EGFR1. (A and B) A2780 cells were transfected with either RCP^wt or the dominant-negative RCP^621E and incubated for 24 h ±1 μM cilengitide for the last 16 h. Adhesion to FN was analyzed using the spinning disc. (A) The analysis of cell density at 61 points for each treatment as a function of wall shear stress (τ₅₀) is shown. The associated curve fit (solid lines) R² values for the fits are 0.95 (RCP^wt with cilengitide) and 0.92 (RCP^621E with cilengitide). The curve fits were used to extract a mean shear stress for cell detachment (τ₅₀) that is proportional to the number of adhesive integrin–ligand bonds (Boettiger, 2007). (B) The combined analyses for τ₅₀ from three independent experiments run in triplicate are shown; normalized percentages are on the bars. Values are mean ± SEM (n = 8 or 9). (C) Cells were transfected with hairpin vectors targeting β3 integrin, α5 integrin, or EGFR1 in combination with GFP, GFP-RCP^wt, or GFP-RCP^621E (left), or were left untransfected (right). After incubation with 1 μM cilengitide (CIL) or 0.5 μg/ml osteopontin (OSP), cells were lysed and GFP-RCP or α5β1 was immunoprecipitated from the lysates using monoclonal antibodies recognizing GFP (anti-GFP), α5 integrin (anti-α5), or an isotype-matched control antibody (RG-16). The presence of α5 integrin, GFP-RCP, endogenous RCP, and EGFR1 in the immunoprecipitates was then detected by immunoblotting. (D–F) A2780 cells were transfected with a short hairpin–targeting β3 integrin (shRNA-β3; F) or were left untransfected (D and E). Confluent monolayers were wounded and the cells were allowed to migrate into the wound in the absence (D and F) or presence (E) of 1 μM cilengitide. The distribution of EGFR1 (green), RCP (red), and F-actin (blue) was visualized by immunofluorescence followed by confocal microscopy. Bar, 30 μm.