Figure 1. Inhibition of αvβ3 promotes α5β1 recycling and association of RCP with α5β1. (A) A2780 cells were transfected with a nontargeting shRNA (sh-CON) or a short hairpin–targeting β3 integrin (sh-β3; right) or were left untransfected (left). Cells were surface labeled with 0.2 mg/ml NHS-S-S-Biotin at 4°C and internalization was then allowed to proceed for 30 min at 37°C. Biotin remaining at the cell surface was removed by exposure to MesNa at 4°C, and internalized integrin was chased back to the cell surface at 37°C for the indicated times in the absence (BAS) or presence (CIL) of 1 μM cilengitide, 1 μM c-RADfV, 4.4 μM cRGDfV, or 0.5 μg/ml soluble osteopontin. Cells were then reexposed to MesNa and biotinylated integrin was determined by capture ELISA using microtitre wells coated with anti-human α5 integrin monoclonal antibodies. The proportion of integrin recycled to the plasma membrane is expressed as a percentage of the pool of integrin labeled during the internalization period. (left) Values are mean ± SEM; n = 7 (BAS), n = 4 (CIL), and n = 3 (cRADfV, cRGDfV, and OSP) independent experiments. (right) Values are mean ± SEM; n = 3 independent experiments. (B) A2780 cells were transfected with plasmids encoding GFP or GFP fused to the class I FIPs (GFP-RCP, GFP-FIP2, and GFP-Rip11). Transfected cells were incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide for 20 min and lysed in a buffer containing 0.15% Tween-20. GFP and GFP fusion proteins were immunoprecipitated from lysates by incubation with beads coupled to a monoclonal antibody recognizing GFP, and these immunoprecipitates analyzed for the presence of α5β1 and αvβ3 by immunoblotting with antibodies recognizing the integrin α5 and β3 chains, respectively. The loading of GFP and GFP fusions was confirmed by immunoblotting with an antibody recognizing GFP (bottom). (C) Cells were transfected with GFP-RCP (top) or left untransfected (bottom), and then incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide for 20 min. Lysates were immunoprecipitated using monoclonal antibodies recognizing α5β1 integrin (anti-α5), αvβ3 integrin (anti-β3), or an isotype-matched control antibody (RG-16). The presence of α5 and β3 integrin, GFP-RCP, and endogenous RCP were detected by immunoblotting. (D) After cotransfection of GFP or GFP-RCP with either nontargeting shRNA (shRNA-CON) or a short hairpin targeting β3 integrin (shRNA-β3), A2780 cells were incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide or 0.5 μg/ml osteopontin (OSP) for 20 min. Cells were lysed and anti-GFP immunoprecipitates probed for the presence of α5β1 and GFP as in B. (E) A2780 cells were transfected with GFP, GFP-RCPwt, or GFP-RCP621E, treated with cilengitide, and then lysed. Anti-GFP immunoprecipitates were probed for α5β1 and GFP as in B.
Figure 1.

Inhibition of αvβ3 promotes α5β1 recycling and association of RCP with α5β1. (A) A2780 cells were transfected with a nontargeting shRNA (sh-CON) or a short hairpin–targeting β3 integrin (sh-β3; right) or were left untransfected (left). Cells were surface labeled with 0.2 mg/ml NHS-S-S-Biotin at 4°C and internalization was then allowed to proceed for 30 min at 37°C. Biotin remaining at the cell surface was removed by exposure to MesNa at 4°C, and internalized integrin was chased back to the cell surface at 37°C for the indicated times in the absence (BAS) or presence (CIL) of 1 μM cilengitide, 1 μM c-RADfV, 4.4 μM cRGDfV, or 0.5 μg/ml soluble osteopontin. Cells were then reexposed to MesNa and biotinylated integrin was determined by capture ELISA using microtitre wells coated with anti-human α5 integrin monoclonal antibodies. The proportion of integrin recycled to the plasma membrane is expressed as a percentage of the pool of integrin labeled during the internalization period. (left) Values are mean ± SEM; n = 7 (BAS), n = 4 (CIL), and n = 3 (cRADfV, cRGDfV, and OSP) independent experiments. (right) Values are mean ± SEM; n = 3 independent experiments. (B) A2780 cells were transfected with plasmids encoding GFP or GFP fused to the class I FIPs (GFP-RCP, GFP-FIP2, and GFP-Rip11). Transfected cells were incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide for 20 min and lysed in a buffer containing 0.15% Tween-20. GFP and GFP fusion proteins were immunoprecipitated from lysates by incubation with beads coupled to a monoclonal antibody recognizing GFP, and these immunoprecipitates analyzed for the presence of α5β1 and αvβ3 by immunoblotting with antibodies recognizing the integrin α5 and β3 chains, respectively. The loading of GFP and GFP fusions was confirmed by immunoblotting with an antibody recognizing GFP (bottom). (C) Cells were transfected with GFP-RCP (top) or left untransfected (bottom), and then incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide for 20 min. Lysates were immunoprecipitated using monoclonal antibodies recognizing α5β1 integrin (anti-α5), αvβ3 integrin (anti-β3), or an isotype-matched control antibody (RG-16). The presence of α5 and β3 integrin, GFP-RCP, and endogenous RCP were detected by immunoblotting. (D) After cotransfection of GFP or GFP-RCP with either nontargeting shRNA (shRNA-CON) or a short hairpin targeting β3 integrin (shRNA-β3), A2780 cells were incubated in the absence (BAS) or presence (CIL) of 1 μM cilengitide or 0.5 μg/ml osteopontin (OSP) for 20 min. Cells were lysed and anti-GFP immunoprecipitates probed for the presence of α5β1 and GFP as in B. (E) A2780 cells were transfected with GFP, GFP-RCPwt, or GFP-RCP621E, treated with cilengitide, and then lysed. Anti-GFP immunoprecipitates were probed for α5β1 and GFP as in B.

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