Grb-2 protein peptide polarization binding assay

Figure 1: Schematic of Grb-2 competition binding assay.

Grb-2 is an adaptor protein which provides a link between growth factor receptors (which become autophosphorylated following ligand binding) and activation of ras which leads to subsequent downstream signaling events(1,2). The Grb-2 protein contains one SH2 domain flanked by two SH3 domains; the SH2 domain binds to phosphotyrosine containing sequences on the C-terminal ends of epidermal growth factor (EGF) and other receptors. In this assay, peptides based on the sequence around pY1068 of EGF receptor were selected(3) and their interaction with Grb-2 investigated.

The polarization assay comprises a fluor labeled phosphopeptide ligand which specifically binds to the Grb-2 protein. The binding results in an increase in the polarization signal when compared to that of the ligand alone. The high polarisation signal generated when the components are bound together can be disrupted with an excess of unlabeled peptide ligands. (Figure 1).

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Methods

Direct binding assays were carried out in black opaque 96-well (maximum working volume 300 ml) microtitre plates (Dynex Technologies Inc., Chantilly, Virginia, USA) by adding 25 ml either a fluorescein labeled 10-mer phosphopeptide ligand or Cy™5- or Cy3B-labeled 9-mer phosphopeptide ligands (final assay concentrations 20 nM) to 20 mM MOPS buffer, pH 7.4 containing 10 mM DTT and 0.005% Tween™ 20. Varying amounts of Grb2 protein (as shown) was added to wells in 50 ml assay buffer. Assays, in a total volume of 150 ml, were incubated for 60 min at room temperature prior to determination in a Jolley Research and Consulting Fluorolite FPM-2™ reader. Filters for fluorescein were excitation 485 nm and emission 535 nm, for Cy5 were excitation 610 nM and emission 690 nM and for Cy3B were excitation 530 nM and emission 590 nM.

Competition assays were performed in 96-well plates (as above) by adding Grb2 protein (1.1 mg in 50 ml assay buffer) and 25 ml of 120 nM preparations of either fluorescein labeled 10-mer or Cy3B-labeled 9-mer phosphopeptide ligands (final assay concentrations 20 nM) to buffer (described above) together with 50 ml varying concentrations of unlabeled phosphorylated competitor peptides (either the 9-mer or a 13-mer). Assays, in a total volume of 150 ml, were incubated for 60 minutes at room temperature prior to determination as described above.

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Results

Fig. 2: Direct Grb-2 phosphopeptide binding assay

Binding of the peptide ligands, labeled with either Cy5 (— ), Cy3B (—) or fluorescein (—), to increasing amounts of the Grb-2 protein are shown in Figure 2. Values are expressed as means + SEM (n=3) and are expressed as specific polarization obtained over that seen in the presence of 100 mM unlabeled phosphopeptide ligand. By analysis of the data using the method of Ohnishi et al (1998), J. Biol. Chem., 273, pp.10210-10215, the KD for the peptides were 333 nM (fluorescein), 250 nM (Cy3B) and 166 nM (Cy5). Each ligand was also assessed as a competitor in an equivalent SPA assay where these observations were also confirmed and found to be of similar affinity to the [3H]13-mer phosphopeptide SPA ligand (approx 400 nM). For reference "start" values for polarization were 62 mP (fluorescein ligand), 105 mP (Cy3B ligand) and 236 mP (Cy5 ligand).

Fig. 3: Grb-2 phosphopeptide competition assays

Competition for binding of the the Grb-2 protein to either Cy3B-labeled ligand in the presence of unlabeled 9-mer (—) or 13-mer (—) or fluorescein labeled ligand in the presence of unlabeled 9-mer (—) or 13-mer (—) competitor peptides is shown in Figure 3. Values are means ± SEM (n=3) and are expressed as %B/Bo derived by standard data analysis. For reference non-phosphorylated peptides gave no competition in the assays. The maximum (Bo) and minimum polarization values for the assays were 205 and 160 mP (Cy3B ligand) and 83 and 52 mP (fluorescein ligand) respectively.

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References

• LOWENSTEIN, E. J., DALY, R.J., BATZER, A. G., LI, W., MARGOLIS, B., LAMMERS, R., ULLRICH, A., SKOLNIK. E. Y., BAR-SAGI, D.
  and SCHLESSINGER. J.
  The SH2 and SH3 domain-containing protein Grb2 links receptor tyrosine kinases to ras signalling.
  Cell, 70, 431-442 (1992).
• CHARDIN, P., CUSSAC, D, MAIGNAN, S. and DUCRUIX, A.
  The GRB2 Adaptor.
  FEBS Letters, 369, 47-51 (1995).
• CUSSAC, D., FRECH, M. and CHARDIN, P.
  Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.
  EMBO Journal, 13, 4011-4021 (1994).