Recent studies have shown that Grb2 mediates not only the canonical Sos/Ras pathway, associated with cell proliferation [Downward, 1994], but also Ras-independent pathways associated with morphological changes [Puto et al., 2003]. the activation of a survival pathway via Bcl-2. The effects of ErbB2 over-expression were abrogated by the humanized anti-ErbB2 monoclonal antibody Herceptin added only from the apical side. The ability of apical ErbB2 to initiate an altered downstream cascade suggests that subcellular localization of the receptor plays an important role in regulating ErbB2 signaling in polarized epithelia. plane (Fig. 1B). In addition, the lateral domains of the transfected cells were not stained although they are positive in permeabilized cells (Fig. 2B), suggesting that ErbB2 over-expression does not grossly affect tight junction competence in these cells. In order to exclude transfection as the cause of ErbB2 relocalization from the basolateral to the apical domain, non-permeabilized cells were transfected with empty vector expressing GFP. The antibodies against the ECD of ErbB2, applied on the apical surface of the unpermeabilized monolayer, showed no apical signal (Fig. 1C). The location of ErbB2 was further addressed by extracellular biotinylation experiments. ErbB2 (+) or mock-transfected (?) Caco-2 confluent cell LY450108 monolayers grown on filters were treated with a non-permeable biotinylating reagent from either the apical or the basolateral side. The cells were then solubilized for affinity purification of the biotinylated cell surface proteins with streptavidin-conjugated agarose. Immunoblotting of the streptavidin precipitates with anti-ErbB2 antibodies demonstrated the surface to which the ErbB2 was exposed. As shown in Figure 1D, ErbB2 was present at the apical surface only in Caco-2 cells transfected with ErbB2 (+), but absent in mock-transfected cells (?). Conversely, the endogenous ErbB2 was found on the basolateral surface in mock-transfected Caco-2 cells (basolateral ?), where it could not be discriminated from the over-expressed molecules in transfected cells because of the large excess of non-transfected cells. Open in a separate window Fig. 1 Relocalization of ErbB2 to the apical surface of polarized Caco-2 cells by over-expression. A: Detection of endogenous ErbB2 on untransfected permeabilized Caco-2 cells. The cells were grown on filters for 10 days and then fixed and challenged with anti-ErbB2 ECD antibody (red). B: Apical localization of transfected (over-expressed) ErbB2. The cells were grown on filters and transfected with ErbB2 4 days after seeding the culture. These confluent non-permeabilized monolayers were fixed and processed with anti-ErbB2 ECD (red channel) from the apical side of the filter. A,B: Single confocal sections in the plane (top panels), and 3D reconstructions from the confocal stack in the aircraft (perpendicular towards the monolayer, apical part up, bottom sections). C: Localization of ErbB2 in polarized mock-transfected Caco-2 cells (bare vector tagged with GFP). The confluent monolayers had been transfected with vector expressing GFP as referred to in (A and B). The set, non-permeabilized monolayer was prepared for immunofluorescence with anti-ErbB2 ECD antibody (reddish colored) just through the apical part of the filtration system. Scale pubs, ACC, 10 m. D: Recognition of apical ErbB2 by extracellular biotinylation in ErbB2- (+) and mock-transfected cells (?). Caco-2 cells had been grown on filter systems, transfected as referred to over and put through biotinylation through the basolateral or apical sides from LY450108 the filtering. Triton LY450108 X-100 components had been pulled-down with streptavidinCagarose as well as the SDS eluates separated by Web page (remaining two lanes of every blot). A complete SDS extract of the transfected monolayer was operate in the right-hand part lane of every blot like a positive control for the antibody (+c). The immunoblot was performed with anti-ErbB2 antibody (best sections), or anti-ErbB3 antibody (bottom level panels). The full total results shown are representative of three independent assays. E: Basolateral localization of ErbB3 in ErbB2-transfected cells by confocal immunofluorescence. Confluent Caco-2 monolayers had been transfected as referred to in (B), and prepared by sequential staining the following: First, staining of non-permeabilized cells was performed with anti-ErbB2 ECD antibody (reddish colored). After that, the cells had been permeabilized and prepared with anti-ErbB3 antibody (green). The pictures are 3D reconstructions ( 0.05). Open up in another windowpane Fig. 2 Apical localization of ErbB2-triggered tyrosine Rabbit polyclonal to PIWIL2 1139 (pY1139) and phosphorylation of p38 in Caco-2 cells. A: Localization by vectorial biotinylation: Caco-2 cells had been transfected with ErbB2 (+) or mock-transfected (?), biotinylated through the apical.