Skip to main content
Figure 3 | BMC Biochemistry

Figure 3

From: The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins

Figure 3

The SH3 domains of Grb2 and Amphiphysin II bind the POB1 338 PPTPPPRP 345 peptide and the mutation R344A squelches the interaction. A). The POB1 PRD1 (P308-V365) region was synthesized as 16 overlapping thirteenmers on a cellulose membrane. The membranes were probed with the SH3 domains of Amphiphysin II (AmphIISH3) or the carboxy-terminal SH3 of Grb2 (Grb2SH3-C) expressed in bacteria as GST fusions. GST alone is used as a negative control. Anti-GST antibodies were utilized to identify bound proteins. The peptide sequences are indicated. The best binding peptides are shown in bold. The three positive control peptides are: GST binding peptides (positive control for GST and secondary antibody) and two peptides containing SH3 interacting motifs. Each binding experiment was carried out in duplicate. The sequence in bold correspond to the best binders. The best peptide 338PPTPPPR344 shows an intensity above 11.000 arbitrary units. Another peptide containing the sequence 214LKARPS219 binds to the SH3 of Amphiphysin, albeit producing a spot intensity around 9000 arbitrary units. Quantitative data are available in additional file 3. B) The POB1 fifteenmer peptide (338PPTPPPRPQKTHSRA352), containing the 338PPTPPPR344 binding motif, was systematically mutagenized by introducing an alanine at each of the indicated 15 positions. The upper membrane was probed with GST as a control, the middle one with the SH3 domain of Amphiphysin II, the bottom one with the carboxy-terminal SH3 of Grb2 fused to GST. The substitution of arginine 344 with alanine reduces the binding of Amphiphysin-SH3 down to 2.5%; a substitution of proline 339 with alanine reduces the binding to 31%. A reduction in Grb2 binding is observed when arginine 344 is substituted with alanine (13%) and when Pro343 or Pro 435 are substituted with Ala (30%). Supplemental quantitative data are provided in additional file 4. C) HEK293 cells were transiently transfected with plasmids expressing GFP fused to the proline rich domain PRD1 of POB1 (lane 1, 2, 3) or with an equivalent construct directing the synthesis of the POB1 PRD1 region mutated in Arg344 (PRD1 R344A), in lanes 4, 5, 6. Cell extracts were adsorbed to glutathione resins containing either the SH3 of Amphiphysin II (top gel) or the C-terminal SH3 of Grb2 (bottom gel). Adsorbed proteins were identified with anti GFP antibodies (for POB1 PRD1). Input is 3% of the lysate utilized in the GST pull down.

Back to article page