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First published online 24 July 2007
doi: 10.1242/jcs.007658

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Mapping ErbB receptors on breast cancer cell membranes during signal transduction

Shujie Yang^1,*, Mary Ann Raymond-Stintz^1,*, Wenxia Ying², Jun Zhang³, Diane S. Lidke¹, Stanly L. Steinberg², Lance Williams³, Janet M. Oliver¹ and Bridget S. Wilson^1,

¹ Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131, USA
² Department of Mathematics and Statistics, University of New Mexico, Albuquerque, NM 87131, USA
³ Department of Computer Science, University of New Mexico, Albuquerque, NM 87131, USA

View larger version (46K): [in this window] [in a new window]   Fig. 1. Biochemical analysis of endogenous ErbB family members in SKBR3 cells. (A) Tyrosine phosphorylation status of ErbB receptors in SKBR3 cells after 2 hours serum starvation (0) or after indicated time course of treatment with either EGF (20 nM) or heregulin (3.2 nM). (B) Kinase activity associated with EGFR or ErbB2 immunoprecipitates prepared from resting, EGF- or heregulin-treated cells, measured in vitro by K-LISA. (C) Effects of kinase inhibitors on EGFR or ErbB2 kinase activity, measured by K-LISA in immunoprecipitates prepared from cell lysates after a 2-minute stimulus with 20 nM EGF. K-LISA values are corrected for baseline color development typical in IgG controls. (D) Co-precipitation assay for ErbB heterodimerization. Where indicated, cells were stimulated with 20 nM EGF or 3.2 nM heregulin, followed by lysis with 1% NP-40 and immunoprecipitation using EGRF-, ErbB2- or ErbB3-specific antibodies. Samples were subjected to SDS-PAGE, followed by immunoblotting with ErbB-specific antibodies. (E). Immunoblotting of equal amount of SKBR3 lysates (10 µg of protein each lane) to document the relative levels of all three ErbB3 family members.

View larger version (88K): [in this window] [in a new window]   Fig. 2. ErbB2 clustering in SKBR3 cells. (A-F) Membrane sheets were prepared from serum-starved cells (A,D,F), from cells pretreated with the combination of 1 µM AG1478 + 20 µM AG879 for 2 hours (B), or from cells that were serum starved, then stimulated with 20 nM EGF for 2 minutes (C,E). Distributions of ErbB2 were determined by immunogold labeling from the inside using RB9040 primary antibodies and 5-nm-gold-conjugated secondary antibodies. In D-F, membranes were also double labeled with 10-nm-gold reagents to detect phosphorylated tyrosine 1248 in the ErbB2 cytoplasmic tail. (G) Hopkins test is positive for ErbB2 clustering (applied to the data from image A). (H) Range of cluster size for resting ErbB2 in five images from the same experiment in 2A. (I) Positive Ripley's test for coincidence of label for pan-specific anti-ErbB2 antibodies vs phospho-specific ErbB2 antibodies. Bars, 0.1 µm.

View larger version (134K): [in this window] [in a new window]   Fig. 3. Analysis of ErbB2 co-clustering with EGFR and ErbB3 in SKBR3 cells. (A-D) Membrane sheets were prepared from serum-starved cells (A,C) or from cells that were serum starved and then stimulated with either 20 nM EGF for 2 minutes (B) or 3.2 nM heregulin for 5 minutes (D). Sheets in A and B were double labeled from the inside with antibodies to ErbB2 (10-nm gold) and EGFR (5-nm gold); Sheets in C and D were double labeled from the inside for ErbB2 (5-nm gold) and ErB3 (10-nm gold). Circles in A-C, as well as the bracket in D, indicate co-clusters of ErbB2 with either EGFR or ErbB3. Labeled arrows point to clusters containing only a single species of ErbB receptor. Bars, 0.1 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 4. Membrane recruitment of Shc following EGF treatment. (A) SKBR3 cells were serum starved (resting), treated for 1 hour with a combination of 1 µM AG1478 + 20 µM AG879, or serum starved and stimulated for 2 minutes with 20 nM EGF or 5 minutes with 3.2 nM heregulin, followed by fractionation to yield crude cytosol and membrane fractions. Samples were subjected to SDS-PAGE and immunoblotting with pan-reactive anti-Shc antibodies. (B,C,E-G) Membrane sheets were prepared from serum starved cells (B,E,F) or EGF-simulated cells (C,G) and either singly labeled with 5 nm gold reagents recognizing Shc or double labeled for Shc and either ErbB2 or EGFR, as indicated by labels on each image. Circles in B,C highlight singly-labeled clusters of Shc in these membranes. Circles in E and F show co-clusters of Shc with ErbB2 or EGFR in serum-starved cells. Bracket in G indicates co-cluster of Shc with EGFR after a 2-minute EGF treatment. (D). Immunoblotting evidence for the co-precipitation of Shc with EGFR and, to a much lesser extent, with ErbB2 and ErbB3. Treatment of cells is indicated. Bars, 0.1 µm.

View larger version (104K): [in this window] [in a new window]   Fig. 5. Redistribution of membrane-associated PI 3-kinase in heregulin-stimulated SKBR3 cells. (A) Cytosol and membrane fractions were prepared from treated and untreated SKBR3 cells, as described in the legend to Fig. 4. Samples were subjected to SDS-PAGE and immunoblotting with anti-p85 antibodies. (B) Immunoblotting evidence for the co-precipitation of p85 with ErbB3 (and very little co-precipation with EGFR or ErbB2). Treatment of cells is indicated. Membrane sheets in C-E were prepared from serum starved cells (C) or cells treated for 5 minutes with 3.2 nm heregulin (D,E); membranes were then double labeled for p85 (5-nm gold) and ErbB3 (10-nm gold). Bold arrows in C and D point to labeled ErbB3 label and fine arrows to labeled p85 label. Circles in E indicate multiple co-clusters of ErbB3 and p85. Bars, 0.1 µm.

View larger version (142K): [in this window] [in a new window]   Fig. 6. Rapid tyrosine phosphorylation of membrane-associated STAT5 after treatment with EGF. (A) Cytosol and membrane fractions were prepared from treated and untreated SKBR3 cells, as described in the legend to Fig. 4. Samples were subjected to SDS-PAGE and immunoblotting with anti-STAT5 antibodies. (B) Immunoblotting evidence for the co-precipitation of STAT5 with EGFR and, to a lessor extent, to ErbB2/ErbB3. Treatment of cells is indicated. (C) Immunoblotting with anti-STAT5 PY694 antibodies demonstrates the time course of STAT5 phosphorylation in response to 20 nM EGF. Membrane sheets in D-F were prepared from serum starved SKBR3 cells (D) or after 2 minutes of treatment with 20 nM EGF (E,F). Sheets in E and D were singly labeled with 5-nm gold reagents that specifically recognize STAT5 when phosphorylated on Tyr694; there is marked increase in the amount of phospho-STAT5 (encircled) but not in the general pool of STAT5 after EGF treatment. The membrane sheet in F was double labeled for phospho-STAT5 (5-nm gold) and EGFR (10-nm gold). Boxed areas mark the few co-clusters of EGFR and phospho-STAT5 in EGF-treated membranes. Bars, 0.1 µm.

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