The insulin receptor (IR) is a heterotetramer constituted of two extracellular α-subunits that bind to the hormone and two β-subunits that possess an intracellular tyrosine kinase (TK) activity. Binding of insulin to the IR results in autophosphorylation of each β-subunit on at least six different tyrosines. This autophosphorylation occurs first on three tyrosines located in the activation loop of the kinase domain (Y1158, 1162 and 1163), resulting in the stabilization of the kinase in an active conformation. Phosphorylations then occur on a tyrosine located in the juxtamembrane region (Y972) and on two tyrosines located in the carboxy-terminal region (Y1328, 1334). Whereas the precise role of the tyrosines located in the C-terminal domain remains debatable, Y972 seems to be a docking site for substrates such as IRS-1 and Shc (Combettes-Souverain & Issad, 1998). Tyrosine phosphorylation of intracellular substrates has an important role in the activation of signalling pathways (mitogen-activated protein (MAP) kinases, phosphatidylinositol 3-kinase (PI3-K)). After activation, the IR is internalized as a tyrosine-phosphorylated fully active kinase, which may still transmit signals at intracellular sites. Termination of the signal involves inactivation of the IR by dephosphorylation of the three tyrosines of the kinase domain (Tonks, 2003). PTP1B is a protein tyrosine phosphatase located in the endoplasmic reticulum that has an important role in the dephosphorylation of these tyrosines after internalization of the IR (Boute et al, 2003). Although dephosphorylation is clearly a crucial mode of regulation, other mechanisms also seem to be important in the control of the activity of TK receptors. Grb14 belongs to the Grb7-like family of adaptor proteins, thus far made up of Grb7, Grb10 and Grb14 (Daly, 1998). These adaptors seem to regulate positively or negatively signalling pathways elicited by different growth factor receptors. Several lines of evidence indicate that Grb14 has a negative role in the regulation of insulin signalling. However, the mechanism by which Grb14 inhibits signalling through the IR remains elusive. In vitro experiments have shown that Grb14 impairs the TK activity of the IR towards exogenous substrates but protects the tyrosine-phosphorylated IR from dephosphorylation by PTP1B (Bereziat et al, 2002). Paradoxical effects of Grb14 on the tyrosine phosphorylation and TK activity of the IR were observed in Chinese hamster ovary (CHO) cells, in which Grb14 either seems to have no effect (Kasus-Jacobi et al, 1998; Hemming et al, 2001) or increases the tyrosine phosphorylation of the IR (Bereziat et al, 2002), while inhibiting the effect of insulin on IRS-1 phosphorylation (Kasus-Jacobi et al, 1998; Hemming et al, 2001), activation of the extracellular signal-regulated kinase (ERK) pathway (Bereziat et al, 2002) and glycogen and DNA synthesis (Kasus-Jacobi et al, 1998). Definitive evidence for a negative role of Grb14 in insulin signalling was obtained with Grb14 knockout mice, which showed improved glucose tolerance and insulin sensitivity (Cooney et al, 2004). However, paradoxical effects were also observed, including a decrease in IR tyrosine phosphorylation associated with an increased tyrosine phosphorylation of IRS-1 in the liver of knockout mice. The decreased IR phosphorylation was attributed to removal of a potentially protective effect of Grb14 on dephosphorylation of the IR by PTP1B. However, the concomitant increase in the tyrosine phosphorylation of IRS-1 observed in the same tissue remained difficult to explain.

The bioluminescence resonance energy transfer (BRET) technique has recently emerged as an important tool for the study of protein–protein interactions in living cells. We previously used this technique to study the interaction of the IR with PTP1B (Boute et al, 2003). In the present work, we use BRET to study, in living cells, the relationships between IR, Grb14 and PTP1B. We also show that, in cells transfected with PTP1B, Grb14 selectively protects the three tyrosines of the activation loop from dephosphorylation, while favouring the dephosphorylation of the tyrosine of the juxtamembrane domain of the IR.

Figure 1 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 (more ...)

Fig 1B,C shows BRET signal between Grb14–YFP and either IR–Rluc or a kinase-dead mutant of the IR fused to luciferase (IR-K1030A–RLuc). Whereas insulin markedly increased BRET between IR–Rluc and Grb14–YFP, it had no effect on BRET between IR-K1030A–Rluc and Grb14–YFP. This result demonstrates that binding of insulin has no effect by itself, and that autophosphorylation of the IR is necessary to induce the Grb14–IR interaction. The specificity of insulin-induced BRET signal measured in these experiments was demonstrated by the low BRET signal obtained in cells expressing IR–Rluc and YFP alone (supplementary information 2 online).

Figure 2 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 (more ...)

Figure 3 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 (more ...)

Figure 4 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 (more ...)

Recruitment of IRS-1 to the activated IR transduces signals that result in the activation of intracellular signalling pathways, including the ERK1/2 (Myers et al, 1994) and the protein kinase B (PKB) pathways (Sun et al, 1993). We (unpublished observations) and others (Shin et al, 2002) found that the PKB pathway is constitutively activated in HEK293 cells. We therefore studied the consequences of Grb14 expression on ERK1/2 activation in cells co-transfected with IR–YFP, IRS-1, PTP1B-wt and Grb14. As shown in Fig 4B, phosphorylation of ERK1/2 was reduced in the presence of Grb14, confirming that the reduction of Y972 phosphorylation had consequences not only on the recruitment of IRS-1 but also on downstream signalling pathways. Although the activation of PKB cannot be evaluated in HEK cells, we observed that Grb14 markedly inhibits the phosphorylation of IRS1 on Y612, which is involved in the binding of PI3-K and activation of PKB (supplementary information 5 online).

Grb10 has also been reported to inhibit the interaction of IRS-1/2 with the IR, resulting in impaired downstream insulin signalling (Wick et al, 2003). However, this inhibitory effect was attributed to physical disruption of the IR/IRS interaction rather than to dephosphorylation of Y972. Differences in the molecular mechanism of binding of Grb10 and Grb14 to the IR have been described, particularly with regard to the respective roles of their SH2 (Src homology 2) and BPS (between Pleckstrin homology and SH2) domains. Indeed, whereas Grb14 is believed to bind to the IR by engaging both its SH2 and BPS domains on the three phosphotyrosines of the activation loop (Depetris et al, 2005), Grb10 is likely to bind to pY972 through its SH2 domain (Frantz et al, 1997) and to the activation loop through its BPS domain (Depetris et al, 2005). These differences may have a role in the different mechanisms by which Grb10 and Grb14 regulate IR/IRS interaction. In agreement with this notion, overexpression of Grb10 does not decrease the phosphorylation of Y972 of the IR in CHO cells, and even seems to increase it (Wick et al, 2003).

To determine whether the effects of Grb14 on site-specific dephosphorylation of the IR could also be detected in a more relevant cellular system, we studied the effect of increasing amounts of transfected Grb14 on the endogenous IR in the human liver cell line HuH7. We observed that increasing the amount of Grb14 specifically decreases the amount of pY972. The lowest amount of transfected Grb14 had no apparent effect on the phosphorylation of the activation loop, and at higher amounts, a slight but not significant decrease was observed. Densitometric analysis of three independent experiments indicates that increasing Grb14 amount by 2.0±0.2-fold (an increase found in tissues of rodent models of insulin resistance and human type II diabetic patients; Cariou et al, 2004) significantly decreased the pY⁹⁷²/pY^{1158,1162,1163} ratio. Increasing the amount of Grb14 by 5.0±0.9-fold further decreased the pY⁹⁷²/pY^{1158,1162,1163} ratio (Fig 5A,B). At these higher levels of Grb14 expression, a decrease in insulin-induced PKB phosphorylation was also observed, indicating that Grb14 also affects the PI3-K/PKB pathway in insulin target cells (Fig 5C).

Figure 5 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 (more ...)

Therefore, a site-specific dephosphorylation of the endogenous IR, associated with inhibition of downstream insulin signalling, is also observed when Grb14 expression increases in human liver cells. Although care should be taken when extrapolating results from cultured transfected cells, this result strongly suggests that our observations may be relevant to pathophysiological situations of insulin resistance.

Altogether, our work suggests that Grb14 binding to the activated IR modifies the interaction of IR with PTP1B. This impairs the dephosphorylation of the three tyrosines of the activation loop by PTP1B, while favouring the dephosphorylation of tyrosine 972. As a recent structural study suggested that Grb14 may act as a pseudosubstrate inhibitor for the IR kinase (Depetris et al, 2005), it is also possible that an inhibition of the IR catalytic activity leads to the decreased phosphorylation of tyrosine 972. This site-specific regulation of the tyrosine phosphorylation of the IR may explain some of the paradoxical effects of Grb14 observed previously. In addition, our results clearly demonstrate that considering the global phosphotyrosine content of the IR as an index of its signalling capability may be misleading, as the phosphorylation of different domains can be regulated in opposite ways.

Materials. All materials have been described previously (Boute et al, 2003), except anti-IRS-1 (UBI, Charlottesville, CA, USA), anti-pERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Erk2 (UBI), anti-pY⁹⁷² and anti-pY^{1158,1162,1163} (Biosource International, Camarillo, CA, USA) and anti-pPKB (Cell Signaling Technology, Danvers, MA, USA) antibodies.

Expression vectors. IR–Rluc, IR–YFP, YFP–PTP1B-wt and YFP–PTP1B-D181A complementary DNAs have been described previously (Boute et al, 2003). Rat Grb14 coding sequence was inserted into pEYFP-N1 (Clontech, Mountain View, CA, USA).

BRET experiments. HEK293 cells were transfected exactly as described previously (Boute et al, 2003) using 300 ng of each cDNA construct, unless otherwise stated in the figure legend. One day after transfection, cells were transferred into 96-well microplates. All BRET measurements were carried out in these plates the following day, and results were expressed in mBRET units (mBU), as described previously (Boute et al, 2003).

Analysis of IR phosphorylation sites. HEK cells were transfected in 35 mm dishes 48 h before the experiments. After insulin stimulation, IRs were partly purified on WGL agarose or immunoprecipitated with anti-GFP antibody (Lacasa et al, 2005) in buffer containing 1 mM of freshly dissolved orthovanadate.

Statistical analysis. Statistical analysis was carried out using a Student's t-test for paired values.

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

We thank J.-F. Tanti for IRS-1 cDNA. This work was supported by the Institut Servier, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer (Comité de Paris) and the Ministère de la Recherche (ACI no. 02 2 0537/8).