Grb14 reduces insulin-stimulated association between IR and IRS-1. Human embryonic kidney cells were co-transfected with IR–YFP, IRS-1 and PTP1B, with or without Grb14. Cells were stimulated with 100 nM insulin for 10 min and lysed. (A) The IR was immunoprecipitated with an anti-green fluorescent protein (GFP) antibody. The amount of IRS-1 co-precipitated with the IR was evaluated with an anti-IRS-1 antibody. (B) Crude cell extracts from the same cells were submitted to western blotting and immunodetected with anti-IRS-1, 4G10 (general anti-phosphotyrosine), anti-pY^{1158,1162,1163}, anti-pY⁹⁷², anti-pERK1/2 and anti-ERK2 antibodies. ERK, extracellular signal-regulated kinase; IR, insulin receptor; YFP, yellow fluorescent protein.

Dynamics of interaction between the insulin receptor and Grb14 in intact living cells. (A) Bioluminescence resonance energy transfer (BRET) was monitored in human embryonic kidney cells coexpressing IR–Rluc and Grb14–YFP. Cells were preincubated for 15 min in the presence of coelenterazine (5 μM) and then stimulated with 5 or 100 nM of insulin. IR, insulin receptor; YFP, yellow fluorescent protein. (B) Interaction of Grb14 with the wild-type and kinase-dead mutant. (C) Basal and insulin-stimulated BRET at time 20 min. Results are means±s.e.m. of seven (IR–Rluc) or two (IR–Rluc-KD) independent experiments (^***P<0.001 when compared with basal condition).

Effect of Grb14 in liver-derived HuH7 cells. HuH7 cells transfected with different amounts of Grb14 complementary DNA were stimulated or not stimulated with 100 nM insulin for 10 min and lysed. (A) After partial purification of insulin receptor (IR) on wheat germ lectin, the phosphorylation of the IR was evaluated by western blotting using site-specific phospho-antibodies (anti-pY^{1158,1162,1163} and anti-pY⁹⁷²). The amount of receptors loaded in each lane was evaluated using an anti-IRβ antibody. (B) After densitometric analysis of the anti-pY^{1158,1162,1163} and anti-pY⁹⁷² phosphotyrosine signals, the pY⁹⁷²/pY^{1158,1162,1163} ratio was calculated. The data presented are means±s.e.m. of three independent experiments (^*P<0.05; ^**P<0.01). (C) Cell extracts were submitted to western blotting. The effect of insulin on the phosphorylation of protein kinase B (PKB) was evaluated using an anti-pPKB antibody. The expression levels of Grb14 and PTP1B proteins in these cells are also shown.

Grb14 regulates the dephosphorylation of the IR in a site-specific manner. Human embryonic kidney cells co-transfected with IR–Rluc alone or in combination with YFP–PTP1B-wt and Grb14 were stimulated or not stimulated with 100 nM insulin for 10 min and lysed. (A) The phosphorylation of IR partially purified on wheat germ lectin was evaluated by western blotting using a general anti-phosphotyrosine antibody (4G10), or site-specific phospho-antibodies (anti-pY^{1158,1162,1163} and anti-pY⁹⁷²). The amount of receptors loaded in each lane was evaluated using an anti-IRβ antibody. Results are representative of five independent experiments. (B) Densitometric analysis of the anti-phosphotyrosine signals corrected for the anti-IR signal. The data presented are means±s.e.m. of three to four independent experiments (^*P<0.05; ^**P<0.01). IR, insulin receptor; NS, not significant; YFP, yellow fluorescent protein.

Grb14 inhibited bioluminescence resonance energy transfer between the insulin receptor and PTP1B. (A) Human embryonic kidney cells were co-transfected with IR–Rluc and YFP–PTP1B-D181A, and either Grb14 or empty vector (pcDNA3). Bioluminescence resonance energy transfer (BRET) was monitored during 20 min in the absence or presence of 100 nM insulin. IR, insulin receptor; YFP, yellow fluorescent protein. (B) Insulin-induced BRET (increase BRET above basal) at time 20 min. Results are means±s.e.m. of three independent experiments and are expressed as a percentage of control cells (cells not transfected with Grb14; ^***P<0.001). (C) YFP–PTP1B-D181A fluorescence and IR–Rluc luminescence as a percentage of control cells. (D) HEK cells were co-transfected with IR and YFP–PTP1B-D181A, and either Grb14 or empty vector (pcDNA3). Cells were stimulated or not stimulated with 100 nM insulin for 10 min and lysed. YFP–PTP1B-D181A was immunoprecipitated with an anti-green fluorescent protein (GFP) antibody. The amount of IR co-precipitated with YFP–PTP1B-D181A was evaluated with an anti-IRβ antibody. (E) Densitometric analysis of IRβ signals. The data presented are means±s.e.m. of three independent experiments. NS, not significant.