Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Mar-Apr;6(2):102-12.
doi: 10.4161/cam.19620. Epub 2012 Mar 1.

Profiling Eph receptor expression in cells and tissues: a targeted mass spectrometry approach

Roberta Noberini  1 , Elena Rubio de la Torre, Elena B Pasquale
Affiliations

Profiling Eph receptor expression in cells and tissues: a targeted mass spectrometry approach

Roberta Noberini et al. Cell Adh Migr. 2012 Mar-Apr.

Abstract

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Binding interactions of ephrin Fc fusion proteins with Eph receptors. Apparent dissociation constant (KD) values were obtained from curves measuring the binding of biotinylated ephrin Fc proteins to Eph receptor Fc proteins immobilized on ELISA plates. h, human; m, mouse; r, rat; nd, not determined.
Figure 2.
Figure 2.
Identification of Eph receptors expressed in PC3 cells. (A) EphA2 and EphB4 were pulled down from PC3 prostate cancer cells by using the indicated ephrin Fc proteins and detected by immunoblot analysis. (B) Schematic representation of the EphA2 peptide coverage obtained from the 1 dimensional LC/MS/MS analysis of EphA2 pulled down with ephrin-A1 Fc in experiment #1 in (C). The extracellular, transmembrane (tm) and cytoplasmic regions of EphA2 are indicated. The peptides are represented as black lines with thickness proportional to the number of peptides with the same sequence identified in the sample (spectral counts). (C) The Eph receptors expressed in PC3 cells were pulled down with the indicated ephrin Fc proteins and identified by 1 dimensional LC/MS/MS analysis. The histograms show the spectral counts obtained for each Eph receptor, with the dark bottom portion of the bars representing the spectral counts that were assigned only to the indicated Eph receptor and the light top portion of the bars representing the spectral counts that could also correspond to other Eph receptors identified in the same experiment. The percentage of sequence coverage by all the peptides identified for each receptor is indicated above the bars. Three independent experiments are shown for ephrin-A1; the ephrin-A1 #3, ephrin-A5 and ephrin-B2 pull-downs were analyzed in parallel in the same experiment.
Figure 3.
Figure 3.
Identification of Eph receptors expressed in the H460 and A549 lung cancer cell lines. (A) The Eph receptors expressed in H460 lung cancer cells were pulled down by using the indicated ephrin Fc fusion proteins, or mixtures of fusion proteins, and detected by immunoblot analysis with the indicated antibodies. (B and C) H460 or A549 cell lysates were subjected to pull-downs with a mixture of ephrin-A1 Fc, ephrin-A4 Fc, ephrin-A5 Fc and ephrin-B2 Fc and the associated Eph receptors were identified by 1-dimensional LC/MS/MS analysis. The histograms show the spectral counts obtained for each Eph receptor, with the dark bottom portion of the bars representing the spectral counts that were assigned only to the indicated Eph receptor. The percentage of sequence coverage for each receptor is indicated above the bars.
Figure 4.
Figure 4.
Identification of Eph receptors expressed in PC3M-luc-C6 tumor xenografts. (A) PC3M xenograft tissue lysed in Triton X-100-containing buffer or modified RIPA buffer was used for pull-downs with a mixture of ephrin-A1 Fc, ephrin-A4 Fc, ephrin-A5 Fc and ephrin-B2 Fc or Fc as a control (indicated by C). Immunoblot analysis reveals similar levels of EphA2 and EphB4 pulled down from the two tumor samples. The two top arrows in the panel showing proteins stained with amido black indicate the ephrin Fc fusion proteins, while the bottom arrow indicates the Fc protein. (B) PC3M-luc-C6 xenografts lysed in RIPA buffer were subjected to pull-down with a mixture of ephrin-A1 Fc, ephrin-A4 Fc, ephrin-A5 Fc (10 μg each) and ephrin-B2 Fc (30 μg each) and the associated Eph receptors were identified by 1 dimensional LC/MS/MS analysis. The histogram shows the spectral counts obtained for each Eph receptor, with the dark bottom portion of the bars representing the spectral counts that were assigned only to the indicated Eph receptor. The percentage of sequence coverage for each receptor is indicated above the bars. (C) Comparison of Eph receptors expression in cultured PC3 and PC3M-luc-C6 cells and in PC3M-luc-C6 tumor xenografts. The PC3M-luc-C6 cells and tumor xenograft lysates were analyzed in duplicate. All samples were lysed in RIPA buffer. (D and E) PC3M-luc-C6 xenografts lysed in RIPA buffer were subjected to pull-down with a mixture of ephrin-A1 Fc, ephrin-A4 Fc, ephrin-A5 Fc (3 μg each) and the associated Eph receptors were identified by 1 dimensional LC/MS/MS analysis as in (B). The histogram in (D) shows the spectral counts obtained for each Eph receptor, as described in (B). The histogram in E shows the spectral counts for EphA2 (for which 13 unique human spectral counts and four unique mouse spectral counts were obtained), EphA3 (for which only spectral counts that could be derived from either the human or mouse receptor were obtained) and EphA5 (for which one unique human spectral count and two unique mouse spectral counts were obtained). The bottom portion of the bars represents the spectral counts that correspond only to the human sequence (green) or mouse sequence (orange) and the top portion of the bars represents the spectral counts that could correspond to either the mouse or the human sequence. The percentage of sequence coverage for each receptor is indicated above the bars. h, human; m, mouse.
Figure 5.
Figure 5.
Identification of Eph receptors that bind ephrin-A3 in mouse hippocampus. (A) Ephrin-A3 Fc or Fc as a control were used for pull-downs from adult mouse hippocampus, which were probed by immunoblotting with anti-EphA4 and anti-Fc antibodies. (B) A mouse hippocampal lysate was subjected to pull-down with ephrin-A3 Fc and the associated Eph receptors were identified by 1 dimensional LC/MS/MS analysis. The histogram shows the spectral counts obtained for each Eph receptor, with the dark bottom portion of the bars representing the spectral counts that were assigned only to the indicated Eph receptor. The percentage of sequence coverage for each receptor is indicated above the bars. (C) Apparent dissociation constant (KD) values obtained from curves measuring ephrin-A3 AP binding to Eph receptor Fc proteins immobilized on ELISA plates; the value for EphA4 is approximate since the ephrin-A3 AP concentration was insufficient to reach maximal binding.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008;133:38–52. doi: 10.1016/j.cell.2008.03.011. - DOI - PubMed
    1. Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer. 2010;10:165–80. doi: 10.1038/nrc2806. - DOI - PMC - PubMed
    1. Pasquale EB. Eph receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol. 2005;6:462–75. doi: 10.1038/nrm1662. - DOI - PubMed
    1. Pasquale EB. Eph-ephrin promiscuity is now crystal clear. Nat Neurosci. 2004;7:417–8. doi: 10.1038/nn0504-417. - DOI - PubMed
    1. Flanagan JG. Neural map specification by gradients. Curr Opin Neurobiol. 2006;16:59–66. doi: 10.1016/j.conb.2006.01.010. - DOI - PubMed

Publication types