Role of ARM domains and PDZ binding site in NPRAP recruitment of ABP to adherens junctions in MDCK cells. NPRAP, ABP-L–GFP, and their mutants were expressed by transfection in MDCK cells as indicated. NPRAP formed a complex with and localized with E-cadherin at adherens junctions, dependent on the NPRAP ARM domain, which is absent in NPRAPΔARM. NPRAP recruited ABP to E-cadherin dependent on the NPRAP PDZ binding site, which is deleted in NPRAPΔ3. The mutant NPRAPΔARMΔ3 lacks both the ARM domains and the PDZ binding site. NPRAP formed a complex with ABP and recruited ABP to E-cadherin at adherens junctions dependent on ABP-Set_I, which is present in GFP–ABP-Set_I and absent in ABP-SΔS_I–GFP. A, Endogenous E-cadherin was immunoprecipitated from extracts of transfected MDCK cells, and the presence of NPRAP or its mutants was assayed by Western blotting. E-cadherin coimmunoprecipitated NPRAP and NPRAPΔ3, but not NPRAPΔARM or NPRAPΔ3ΔARM, indicating that the ARM domain of NPRAP but not the PDZ binding site is necessary for NPRAP association with E-cadherin. B, NPRAP was immunoprecipitated from extracts of transfected MDCK cells, and the presence of E-cadherin and ABP-L–GFP was assayed by Western blotting. NPRAP associates with both ABP-L–GFP and E-cadherin. The NPRAP PDZ binding site and the ARM repeats are necessary for binding, respectively, to ABP-L–GFP and E-cadherin. C, ABP-L–GFP and NPRAP or its mutants were visualized by immunofluorescence in transfected MDCK cells. i, ii, NPRAP deletion mutants lacking the ARM repeats, NPRAPΔARM and NPRAPΔARMΔ3, do not colocalize with E-cadherin and are diffuse throughout the cytoplasm. iii, NPRAPΔARM colocalizes with ABP-L–GFP in the cytoplasm, but not with E-cadherin, which is localized to the adherens junction. iv, NPRAPΔARMΔ3 does not colocalize with ABP-L–GFP or E-cadherin. D, ABP-L–GFP and NPRAP or its mutants were expressed by transfection in MDCK cells. NPRAP, ABP-L–GFP, and endogenous E-cadherin were visualized by immunofluorescence. i, ABP lacking Set_I, ABP-SΔS_I–GFP, forms cytoplasmic clusters and does not colocalize with E-cadherin, which is at adherens junctions. ii, ABP-SΔS_I–GFP does not colocalize with NPRAP or E-cadherin in MDCK cells. iii, GFP–ABP-Set_I, when transfected on its own into MDCK cells, is diffusely distributed in the cytoplasm and does not colocalize with E-cadherin. iv, When GFP–ABP-S_I and NPRAP are cotransfected into MDCK cells, the two colocalize with each other and with E-cadherin at adherens junctions. E, Set_I of ABP is necessary and sufficient for ABP to associate with NPRAP in MDCK cells. After cotransfection with NPRAP, GFP–ABP-Set_I and ABP-L–GFP coimmunoprecipitated with NPRAP from cell extracts, whereas ABP-SΔS_I–GFP did not coimmunoprecipitate with NPRAP. IB, Immunoblot; IP, immunoprecipitation.