Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion - PubMed (original) (raw)

Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion

Michael P Sanderson et al. J Cell Biochem. 2008.

Abstract

Exosomes are small membrane vesicles derived from intracellular multivescicular bodies (MVBs) that can undergo constitutive and regulated secretion from cells. Exosomes can also secrete soluble proteins through metalloprotease-dependent ectodomain shedding. In this study, we sought to determine whether ErbB1 receptors are present within exosomes isolated from the human keratinocyte cell line, HaCaT, and whether exosome-associated ErbB1 receptors can undergo further proteolytic processing. We show that full-length transmembrane ErbB1 is secreted in HaCaT exosomes. EGF treatment and calcium flux stimulated the release of phosphorylated ErbB1 in exosomes but only ligand-stimulated release was blocked by the ErbB1 kinase inhibitor, AG1478, indicating that ligand-dependent ErbB1 receptor activation can initiate ErbB1 secretion into exosomes. In addition, other 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 that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor.

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Figures

Fig. 1

Identification of soluble ErbB1 associated with secreted membrane vesicles. A: Equal numbers of HaCaT cells were treated for 2 h in serum-free medium with or without ionomycin (1 μM) or PMA (50 ng/ml). Cell lysates were collected by direct lysis in SDSPAGE sample buffer and then analyzed by Western blot using antibodies against the ErbB1 C-terminus and β-Actin. B: Conditioned media (CM) from HaCaT cells treated with ionomycin or PMA, as above, were collected and membrane vesicles isolated by ultracentrifugation. Vesicles were directly taken up in SDS–PAGE sample buffer and analyzed by Western blot using antibodies against the ErbB1 C-terminus and phospho ErbB1 C-terminus (residue Tyr1068). C: Vesicles from HaCaT CM were isolated as in (B). The vesicle pellet is a mixture of exosome and membrane bleb components [Stoeck et al., 2006]. To separate these components, vesicles were centrifuged on a sucrose step gradient as described in the experimental procedures section. Protein fractions from the gradient were analyzed by Western blotting using the anti-ErbB1 C-terminus antibody and a range of other exosomal marker proteins and disintegrin metalloproteases as indicated. The density of each fraction of the gradient is shown. D: Vesicles from HaCaT cells were collected by ultracentrifugation and then resuspended in PBS in preparation for electron microscopy at 48,000× magnification. Exosomes displayed a disk like structure consistent with the findings of other groups [Andre et al., 2002b; Gill et al., 2005]. E: Isolated vesicles were adsorbed to latex beads and subjected to FACS analysis using an antibody to the ectodomain of ErbB1.

Fig. 1

Fig. 2

Intracellular vesicles containing ErbB1 are released from the cell as exosomes. A: Cell homogenates were layered on a step sucrose gradient to separate intracellular vesicles and plasma membranes. Gradient fractions were analyzed by Western blot using an antiErbB1 C-terminus antibody. In addition, antibodies to marker proteins were used in order to confirm the separation of organelles. EEA1, early endosome antigen 1; BIP/GRP78, binding protein/glucose-regulated protein 78 (endoplasmic reticulum marker); LAMP-1, lysosome associated membrane protein-1; TGN, trans Golgi network; ER, endoplasmic reticulum. Plasma membrane fractions were identified by cell surface biotinylation followed by homogenization and gradient fractionation. Biotinylated proteins were detected with Streptavidin-HRP. Note that ADAM10 has previously been shown to predominantly localize to the Golgi, TGN and intracellular vesicles by cell fractionation and immunohistochemistry [Gutwein et al., 2003]. B: Fractions 3–6 from the cell homogenate gradient in A were pooled and ErbB1 protein fragments analyzed by Western blotting with the anti-ErbB1 C-terminus antibody. In order to visualize less represented smaller fragments, the blot was overexposed (right panel). Fraction 11 of the gradient, representing the plasma membrane (PM) and ER components (left panel) was also analyzed alongside the vesicle fractions.

Fig. 3

Ionomycin leads to ErbB1 trafficking to MVB compartments. A: HaCaT were treated with 1 mM ionomycin for the indicated length of time and cells were analyzed for apoptosis using Annexin-V and PI staining and FACS analysis. Treatment with staurosporine (1 μM) for 16 h served as positive control. B, C: HaCaT cells were treated with ionomycin (1 μM) for 10, 30, and 60 min and cell homogenates layered on a step sucrose gradient as described above. Homogenate gradient fractions and exosomes isolated from CM were analyzed by Western blot using the anti-ErbB1 C-terminus antibody. D: HaCaT cells were grown on glass cover slips and treated with or without ionomycin (1 μM) for 20 min. Cells were fixed and permeabilized in 3% paraformaldehyde/Triton X100 and stained with a mouse anti-ErbB1 ectodomain antibody followed by a Cy3-conjugated antimouse IgG secondary antibody. Confocal images were taken in the DKFZ microscopy division. [Color figure can be viewed in the online issue, which is available at

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Fig. 3

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Fig. 4

EGF activates release of ErbB1 in exosomes. A: HaCaT cells were treated in the presence or absence of EGF (20 ng/ml) and ionomycin-calcium salt (1 μM) for 2 h. Exosomes were isolated from conditioned media by ultracentrifugation and then analyzed by Western blot using antibodies against the ErbB1 C-terminus, phospho-ErbB1 C-terminus and the exosomal marker protein moesin. B: HaCaT cells were pre-treated with or without AG1478 (0.25 μM) for 30 min prior to stimulation with EGF (20 ng/ml). Exosomes were analyzed by Western blot for ErbB1 as in (A). C: HaCaT cells were pre-incubated for 30 min in AG1478 (0.25 μM) or EGTA (10 mM) and then treated with ionomycin (1 μM). Exosomes were then analyzed by Western blot for ErbB1 as in A.

Fig. 5

Characterization of ErbB1 isoforms in exosomes and soluble forms generated by metalloprotease cleavage. A: Exosomes from HaCaT cell CM were pelleted by ultracentrifugation as described above. Following this, soluble non-membrane associated ErbB1 ectodomains in the supernatant were immunoprecipitated using a mouse anti-ErbB1 ectodomain antibody. Exosome and IP samples were analyzed by SDSPAGE and Western blotting for the ErbB1 C-terminus, phospho-ErbB1 C-terminus and N-terminal ErbB1 ectodomain. B: HaCaT cells were treated for 2 h with; serum-free media with DMSO (vehicle), ionomycin (1 μM) or PMA (50 ng/ml; left panel), DMSO or the metalloprotease activator APMA (middle panel), or ionomycin in the presence of DMSO or the metalloprotease inhibitor GM6001 (right panel). Exosomes were removed from the CM by ultracentrifugation and IP performed as described in A. Samples were then analyzed for soluble ErbB1 levels by Western blot analysis with a goat anti-ErbB1 ectodomain antibody.

Fig. 6

ErbB1 ectodomains bind the ligand betacellulin (BTC). Concentrated HaCaT CM free of exosomes was incubated with recombinant BTC and then the ErbB1 ectodomains immunoprecipitated using the mouse anti-ErbB1 ectodomain antibody and protein-A sepharose. As controls, BTC or CM alone were used. Immunoprecipitates were analyzed by Western blot using rabbit anti-BTC and goat anti-ErbB1 ectodomain antibodies.

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Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion - PubMed (original) (raw)