After insulin stimulation, the insulin receptor is downregulated by several mechanisms, including dephosphorylation by protein tyrosine phosphatases such as PTP1B28,29
and binding of the adapter protein Grb147
. The BPS region in Grb14 directly inhibits the catalytic activity of the insulin receptor by binding as a pseudosubstrate in the kinase active site, and the C-terminal SH2 domain binds to the phosphorylated activation loop10
. Our current structural and biochemical studies focused on the RA and PH domains of Grb10 and Grb14, and demonstrated that they, in addition to the BPS region and SH2 domain, are required for inhibition of insulin signaling by Grb14 ().
The crystal structure of Grb10 RA-PH reveals that these two domains, separated in sequence by ~40 residues, nevertheless physically associate to form a single structural unit. The RA-PH interface is conserved in Grb14 and Grb7. We propose that the role of the integrated RA-PH structural unit is to optimize binding of small GTPases (e.g.
, Ras) at the plasma membrane. That is, binding of the PH domain to membrane phosphoinositides will favorably position the structurally coupled RA domain for binding to membrane-anchored GTPases. As judged from the Grb14-Ras-insulin receptor structural model (), the particular mechanism of membrane anchoring of a GTPase—N-terminal (e.g.
, Arf proteins) versus C-terminal (e.g.
, Ras proteins), length of the tether—could dictate whether a given GTPase will interact with an RA-PH unit, in addition to the intrinsic GTPase-RA domain binding affinity. For example, the Rap1 C-terminal tail is seven residues shorter than that of N-Ras, which could explain, in part, why Rap1 binds poorly to Grb14 (data not shown) and Grb726
Figure 6 Model of the interaction between Grb14, Ras and the insulin receptor. The two insulin receptor kinase (IRK) domains are shown in surface representation (opaque) colored dark (N lobe) and light (C lobe) gray. The juxtamembrane regions linking the transmembrane (more ...)
Most PH domains bind to membrane phosphoinositides and do so with modest affinities21
. The measured binding affinities to the Grb10 PH domain are in the 4–10 μM Kd
range (for PIP2
, ), whereas for the Grb14 PH domain, the range is ≥30 μM. The sequences of the Grb10 and Grb14 PH domains, particularly a glutamic acid in the β1-β2 loop (Glu243 in Grb10) (), suggested that these PH domains may bind phosphoinositides non-canonically20
. The mutagenesis data (), although not unequivocal, indicate that the Grb10 PH domain binds phosphoinositides non-canonically, but that the Grb14 PH domain may bind phosphoinositides canonically. Because Grb14 RA-PH showed modest in vitro
specificity toward PI(3,4,5)P3
(), we tested whether wortmannin, an inhibitor of PI-3K, affects the ability of Grb14 to be recruited to the insulin receptor. The co-immunoprecipitation results (data not shown) suggest that PI-3K activity is not required for Grb14 recruitment to the receptor.
Interestingly, PH domains have been implicated in binding directly to activated GTPases30
, raising the question whether the Grb14 PH domain, which binds phosphoinositides weakly, could bind directly to GTPases together with the RA domain. Based on the relative position of the RA and PH domains in the Grb10 RA-PH structure and the mode of GTPase binding to an RA domain18,27
(with corroboration from the Grb14 K140A result ()), a direct interaction between the Grb14 PH domain and a GTPase appears unlikely.
Grb14 binds better to activated Ras than Grb10 (), although it is not clear from the Grb10 RA-PH structure and a sequence comparison of the RA domains why this might be so. Because membrane recruitment via the PH domain is important for the interaction of Grb14 with Ras (K252A in ), differences in the phosphoinositide binding characteristics of the PH domains of Grb10 and Grb14 might account for the differential ability to interact with Ras. It is conceivable that the higher-affinity (and more promiscuous) PH domain of Grb10 results in recruitment to different regions of the plasma membrane than Grb14, or perhaps to internal membranes. Of note, dimerization of the RA-PH unit through the C-terminal extension of the PH domain will increase membrane binding avidity21
. Although Grb10 RA-PH only weakly dimerizes in solution (Supplementary Fig. 4
online), dimerization of the Grb10/14 SH2 domain31
, along with membrane association of the PH domain, will enhance formation of the RA-PH dimer.
Our functional studies monitoring Akt and ERK activation downstream of insulin stimulation demonstrated that Grb14 is a more potent inhibitor of insulin signaling than Grb10 (), at least in CHO-IR cells, which is probably due to more efficient recruitment of Grb14 to the insulin receptor (). Previous biochemical experiments showed that the Grb14 BPS region is a more potent inhibitor (and thus better binder) of the insulin receptor than the Grb10 BPS region32
. Our data demonstrating that Grb10 is a weak binder of Ras compared to Grb14 (), and that GTPase is evidently necessary for full recruitment of Grb14 to the insulin receptor (K140A in ), indicate that the RA-PH unit of Grb14 also contributes to its inhibitory signaling potency versus Grb10. In light of these biochemical results for Grb10, it is somewhat surprising that Grb10−/−
mice display an improved insulin-signaling phenotype4,5
, although the expression levels of Grb10 versus Grb14 in insulin-responsive cells has not been characterized.
The tandem RA-PH domains of Grb7-10-14 are conserved in a second adapter-protein family (MRL proteins11
) comprising the Caenorhabditis elegans
, the mammalian proteins RIAM13,14
and the Drosophila melanogaster
, all of which are Ena/VASP-binding proteins involved in actin-cytoskeleton rearrangement. Genetic evidence indicates that MIG-10 interacts with the small GTPase CED-10/Rac133
. RIAM, although named for its ability to interact with Rap113
, was shown in a different study to bind instead to Ras14
. Despite efforts to do so, no small GTPase has been identified as a lamellipodin binder15
. The PH domains of RIAM and lamellipodin, like Grb10 and Grb14, contain an acidic residue in the β1-β2 loop (Glu243 in Grb10, Supplementary Fig. 2
online), which suggests that these PH domains might bind phosphoinositides via the non-canonical mode. Whether in RA-PH-containing proteins the phosphoinositide binding mode influences GTPase binding or specificity remains to be determined.
In response to insulin, a number of cellular signaling pathways are activated, including the PI-3K and ERK pathways, which are then attenuated at the level of the insulin receptor by PTP1B and Grb14. The mechanisms that govern the kinetics of insulin signaling are not well understood, in particular, how the duration of positive signaling is controlled. Dephosphorylation of the insulin receptor by endoplasmic reticulum-tethered PTP1B is thought to require endocytosis of the receptor, and the time scale for this process (minutes) provides a window of opportunity for recruitment and phosphorylation of IRS proteins to activate the PI-3K and ERK pathways. In contrast, the negative regulator Grb14 is a multi-domain cytoplasmic adapter protein, not unlike the PH-PTB-containing IRS proteins, which binds to the phosphorylated (activated) kinase domain of the insulin receptor. We have shown here that Grb14-mediated inhibition of insulin signaling requires functional RA and PH domains, and that Grb14 can bind to activated Ras. Therefore, an attractive hypothesis is that Ras activation serves as a timing mechanism for the negative-feedback inhibition of insulin signaling by Grb14.