An antibody to the C-terminus of ErbB1 readily detected the full-length 185 kDa ErbB1 receptor in the membrane pellet of ionomycin treated cells (Fig. immunoreactive but truncated ErbB1 isoforms were detected in exosomes suggestive of additional proteolytic processing. We demonstrate that cellular and exosomal ErbB1 receptors can undergo ectodomain shedding to generate soluble N-terminal ectodomains and membrane-associated C-terminal remnant fragments (CTFs). ErbB1 shedding was activated by calcium flux and the metalloprotease activator APMA (4-aminophenylmercuric acetate) and was blocked by a metalloprotease inhibitor (GM6001). Soluble ErbB1 ectodomains shed into conditioned medium retained the ability to bind exogenous ligand. Our results provide new insights into the proteolysis, trafficking and fate of ErbB1 receptors and suggest A-582941 that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor. to remove cellular debris. Membrane vesicles were collected by centrifugation at 100,000for 2.5 h at 4C using a Beckman SW 40 rotor. Vesicles were directly dissolved in SDS sample buffer or processed further for gradient centrifugation as described below. FACS Analysis FACS analysis of isolated vesicles was done after adsorbing of isolated vesicles to 4 mm (Surfactant-free) A-582941 aldehyde-sulfate latex beads (Interfacial Dynamics Corp., Portland, OR) as described [Stoeck et al., 2006]. The staining of beads with mAbs and PE-conjugated secondary antibodies has been described [Stoeck et al., 2006]. The indication of apoptosis was measured using the FITC-Annexin-V kit from BD (BD Bioscience, Heidelberg, Germany). Stained beads or cells were analyzed with a FACScan using Cellquest software (Becton & Dickinson, Heidelberg, Germany). Homogenization of Cells for Membrane Vesicle Preparation HaCaT cells were scraped from the tissue culture plate surface and taken up in 10 ml PBS. Cells were then centrifuged at 300using a Beckman SW40 rotor. Twelve 1 ml fractions were collected from the top of the gradient and proteins precipitated with chloroform/methanol. Samples were analyzed by SDSCPAGE and Western blotting. Cell Surface Biotinylation Cell surface proteins were biotinylated using EZ-Link Sulfo-NHS-Biotin-Reagent (Pierce). After washing cells for 2 times with icecold PBS, cell surface proteins were labeled by incubation with 0.5 mg/ml biotin reagent for 30 A-582941 min at 4C. The reaction was stopped by washing the cells with 100 mM glycine/PBS. Isolation of Shed ErbB1 Ectodomains From CM For constitutive shedding of ErbB1, HaCaT cells were A-582941 cultured for 24 h in serum-free medium. For activated shedding, cells were treated for 2 h using ionomycin (1 M), APMA (50 M) or PMA (50 ng/ml). CM was collected and cellular debris and membrane vesicles removed by centrifugation at 10,000for 20 min and 100,000for 2.5 h, respectively. The cleared supernatant was then incubated overnight at 4C with 2 g of the mouse anti-ErbB1 ectodomain antibody in the presence of complete protease inhibitor cocktail (Roche). Protein A-Sepharose (GE Healthcare, Freiburg, Germany) was then added and the mixture further incubated for 4 h at 4C. Immunoprecipitates were isolated by centrifugation at 10,000and washed twice with ice-cold PBS. Proteins were solubilized by direct addition of SDSCPAGE sample buffer in preparation for Western blot. Biochemical Analysis SDSCPAGE under reducing conditions and transfer of proteins to Immobilon membranes (Millipore) using semi-dry blotting has been described previously [Gutwein et al., 2000, 2003]. After blocking with 5% non-fat milk in TBS, blots were developed with the respective primary antibody followed by peroxidase conjugated secondary antibody and ECL detection. Confocal Immunohistochemistry and Electron Microscopy The staining of cells grown on glass cover slips with primary antibodies and Cy3-conjugated secondary antibodies has been described previously [Mechtersheimer et al., 2001]. Vesicles from HaCaT cells cultured under constitutive conditions for 24 h in A-582941 serum-free DMEM Rabbit Polyclonal to SEPT2 were collected and analyzed using a Zeiss 10 ? electron microscope as previously described [Gutwein et al., 2003]. Binding of ErbB1 Ectodomains to Betacellulin To analyze the ability of shed ErbB1.