Figure 2.

In oocytes deprotected from their follicular cells, the epidermal growth factor receptor (EGFR)–EGF signaling complex recruits integrin β1, phosphorylated Grb7, but lacks OGT. Stage VI Xenopus oocytes, with surrounding follicular cells (FC) or defolliculated (DE), expressing EGFR were injected with 20 ng of Grb7 or not 1 h before EGF stimulation (5 nM) or left unstimulated. A, after protein normalization, Western blots were performed on FC or DE oocytes, and isolated follicular cells (IFs) with anti-integrin β1, anti-FAK, and anti-actin antibodies. B, immunoprecipitations were performed with anti-EGFR or anti-GST-Grb7 antibodies, and Western blots were performed with antibodies against EGFR, phospho-tyrosine (P-Tyr), O-GlcNAcylation (O-GlcNAc), GST (Grb7-GST), O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), FAK, and integrin β1. Control (Ct) was an immunoprecipitation performed with anti-GST-Grb7 on FC cells expressing EGFR and injected with Grb7. C, sWGA pulldowns were performed with or without competition with 0.5 M free GlcNAc and followed by Western blot analysis with anti-O-GlcNAcylation (O-GlcNAc) or anti-GST-Grb7 antibodies. D, after immunoprecipitations with anti-integrin β1, Western blots were performed with anti-EGFR, -GST (Grb7-GST), -OGT, and -integrin β1 antibodies. Ct: control as in B. E, scheme showing Grb7 domains and competitive Grb7 peptides: proline-rich (PR), RalGEF/AF6, or Ras associated like (RA), Pleckstrin homology (PH), phospho-tyrosine interacting region PIR (BPS, between PH and SH2); and Src homology 2 (SH2). F–H, Grb7 peptides Y188, Y338, both peptides (G7-YY), control mismatched peptide (G7-MP), or anti-FAK antibodies were microinjected with Grb7 (20 ng), 1 h before EGF stimulation. Ct: control as in B. Experiments were performed on 25 oocytes and three different females. FAK, focal adhesion kinase; GST, glutathione-S-transferase; sWGA, succinylated wheat germ agglutinin.