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Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 or PIP2] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP2 and other phosphoinositides. Here, we report a novel requirement for PIP2 in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP2 levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP2 levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP2 and PIP2-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP2. Neutralization of positively charged amino acids abolished EGFR/PIP2 interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR autophosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP2 and point to the basic JD’s critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP2 may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 (or PIP2)] is a phospholipid of pivotal importance to eukaryotic membranes. Receptor-induced hydrolysis of PIP2 by phospholipase C (PLC) into the second messengers diacylglycerol and inositol triphosphate and receptor-mediated phosphorylation of PIP2 by phosphatidylinositol 3-kinase (PI3K) into PIP3 initiate two vital receptor-regulated signaling pathways (the PLC and PI3K pathways, respectively), whereas PIP2 itself can bind to an increasing number of effectors, influencing a variety of cellular processes [5, 7]. PIP2 has been implicated in cytoskeletal organization [14, 51], membrane trafficking and vesicle exocytosis, and endocytosis and motility [24, 47]. Decreased nerve terminal PIP2 levels lead to synaptic defects and early postnatal lethality in mice . Most ion channels and transporters tested show dependence of their activities on levels of plasma membrane PIP2 . Examples of PIP2-dependent ion channels include K+ channels, such as the inward rectifiers (Kir)  and the M-current [43, 52], voltage-gated Ca2+ channels , TRP channels [3, 35], and NMDA receptors [23, 26] (for a recent review, see Logothetis et al. ). Extracellular signals acting through G protein-coupled or growth factor receptors that can cause PIP2 hydrolysis, dramatically affect transporter function and ion channel gating . Structure-function studies aiming to identify the channel sites of interaction with PIP2 have revealed a hotspot of interactions in a highly basic region of the juxtamembrane domain (JD) of the protein immediately proximal to the membrane.
The epidermal growth factor receptor (EGFR, also known as ErbB1) is a member of the receptor tyrosine kinase (RTK) family. The EGFR and other ErbB RTKs play crucial roles in cell growth, survival, proliferation, and differentiation as well as carcinogenesis. Mice lacking EGFR die shortly after birth due to multi-organ failure, developing skin, lung, pancreas, and gastrointestinal tract abnormalities and progressive neurodegeneration [27, 40]. Binding of EGF induces dimerization of EGFR monomers, followed by trans-autophosphorylation of key tyrosine residues on the EGFR intracellular tails, which up-regulates their intrinsic RTK activity . The EGFR is functionally coupled to the turnover of PIP2, as direct tyrosine phosphorylation of phospholipase Cγ by activated EGFR triggers PIP2 hydrolysis to IP3 and diacylglycerol, which leads to Ca2+ mobilization and protein kinase C (PKC) activation, respectively [32, 33].
Although studies of effects of EGF on excitable membranes are expanding, the mechanism that links EGF stimulation to the trans-autophosphorylation and activation of the receptor remains elusive. It is however increasingly appreciated that the JD of EGFR plays a central role in the activation process [1, 15, 25, 31, 44]. Recent structural studies coupled with mutagenesis have provided compelling evidence that the distal JD (termed JM-B or JMAD) stabilizes the activated dimmer via interactions revealed by crystal structures [25, 44]. Yet, the precise role of the proximal JD (here referred to as the basic JD) is not clear . Here we use a variety of approaches (i.e., biochemical, genetic, and electrophysiological) to show that EGFR phosphorylation and downstream signaling to ion channels depend on receptor interactions with PIP2. A cluster of positively charged residues in the basic JD of EGFR is likely to be involved in the binding of EGFR to PIP2. Neutralization mutations of the positively charged amino acids abolishes EGFR/PIP2 interaction, suppresses EGF-induced EGFR auto-phosphorylation and down-regulates EGF-induced EGFR signaling to ion channels. Furthermore, pharmacological or genetic down-regulation of PIP2 levels decreases EGF-induced receptor phosphorylation, whereas up-regulation of PIP2 levels augments it. Since EGFR activation leads to hydrolysis of PIP2, we propose that its functional dependence on PIP2 can serve as a mechanism of self-desensitization.
Wortmannin was purchased from Sigma and antibodies were purchased from Cell Signaling Technologies (EGFRP-Y992), Santa Cruz Biotechnologies (EGFR, β-tubulin), Millipore (anti phospho-Tyrosine 4 G10) and Genetex (PIP5KIα).
A Biacore 2000 optical biosensor was used together with Biacore streptavidin-coated (SA) sensor chips and BIA Evaluation software. The SA chips were washed and equilibrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or phosphate-buffered saline (PBS) buffer (pH 8.0, 20 μl/min), Soluble biotinylated C6 PIP2 was purchased from Echelon and 1–10 mM stocks were prepared in water. After brief sonication, biotinylated PIP2 was immobilized by consecutive injections (3 μl/min, 90 min) until an approximate increase of 300 resonance units was achieved. Peptides were introduced at five serial dilutions in Biacore buffer (1 mM DTT, 2.5 mM MgCl2, 20 mM HEPES, 1 mM EDTA, 150 mM NaCl, 0.005% P20) in 60-100 μl injection volumes and at a flow rate of 20 μl/min. Binding was measured at 25°C for 5 min, followed by 15 min of dissociation. Then 1 min of wash with regeneration buffer was performed (1 M NaCl, 12.5 mM NaOH). The whole process was then repeated three times for each dilution sample. Sensorgram data were analyzed with the BIA evaluation kinetics package and the equilibrium affinity constant was calculated. Peptides were at least 95% pure (Biosynthesis).
Human EGFR, Kir3.4*(S143T) (the Kir3.4 mutant that yields large homomeric currents), synaptojanin (a gift from Dr. Pietro DeCamilli, Yale University, CT) and PI(5)P kinase (a gift from Drs. Show-Ling Shyng and Colin Nichols, Washington University, MO) cDNAs were subcloned into the high-expression vector pGEMsH . Point mutations were introduced using the Quikchange mutagenesis kit (Stratagene) and custom primers with nucleotide mismatch (Invitrogen) and were confirmed by sequencing. cRNAs were transcribed using an in vitro transcription kit with the T7 promoter (Ambion).
Stage V or VI Xenopus laevis oocytes were surgically removed from ovaries and digested with collagenase using standard methods . Oocytes were kept for 2–4 days at 17°C. Oocytes were injected (50 nl/oocyte) with full-length wild-type or mutant EGFR (5 ng), Kir3.4*(S143T) (2 ng), synaptojanin (10 ng) or PI(5)P kinase (10 ng) cRNAs, and analyzed 2–3 days post injection.
Electrophysiological recordings were performed 2–4 days after injection using a DAGAN two-electrode voltage-clamp amplifier and currents were obtained with a square pulse protocol. For the measurements of calcium-activated chloride currents, the bath solution (Ringer’s) contained (in mM): 115 NaCl, 2.5 KCl, 1.8 BaCl2, and 10 HEPES, pH 7.2. For the experiments with Kir3.4 (we used the S143T mutant that yields large homomeric currents) the bath solution (high potassium, HK) contained (in mM): 91 KCl, 1 NaCl, 1 MgCl2, and 5 HEPES/KOH. HK supplied with 3–4 mM BaCl2 was added at the end of the experiment to block potassium currents and obtain a measurement of the leak current. Rapid solution exchange was achieved with a gravity flow perfusion system converging on a 6 μl oocyte chamber engineered in our laboratory from inert materials.
Equal numbers of 25-50 oocytes for each experimental group were stimulated with EGF (100 ng/ml for 10 min) in PBS supplied with phosphatase inhibitors (10 mM NaVO4, 10 mM NaF) and directly homogenized in 5 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM EGTA containing 10 mM NaVO4, 10 mM NaF and complete protease inhibitor cocktail (Roche). For preparation of the membrane fraction, homogenates were centrifuged for 5 min at 5,000 RPM (4°C) and supernatants were subsequently ultracentrifuged at 100,000×g for 40 min at 4°C in a Beckman Maxima Ultracentrifuge. The pellets were re-suspended with a suitable volume of lysis buffer (0.5–1 μl per oocyte), mixed with sodium dodecyl sulfate (SDS)-PAGE loading buffer, boiled for 10 min and analyzed by Western blotting. Total EGFR levels in the samples were determined either on the same membrane used for phosphorylated EGFR (after stripping and re-probing) or on duplicate membranes. For the statistical analysis, densitometric data were normalized for EGFR expression levels and expressed relative to the normalized value of the control in each experiment. Results are expressed as mean±SEM. Statistical significance was determined with a Student’s t test.
HeLa cells were plated in 60-mm dishes and serum starved with 0.5% fetal bovine serum for 16 h in the presence of 100 nM or 15 μM Wortmannin, or vehicle DMSO. After 16 h, the cells were stimulated with 100 ng/ml EGF for 10 min, then lysed in 1%SDS solubilization buffer in the presence of protease (Roche) and phosphatase (Sigma) inhibitor cocktails. 50 μg of lysate was analyzed by Western immunoblotting (WB) as described above. For siRNA transfection, HeLa cells were plated and transfected according to the manufacturer’s (NeoFx kit, Ambion) instructions, using siRNA targeting exons 9,10 of human PIP5KIα in a final concentration of 100 nM in OPTIMEM medium. Cells were incubated for 48–72 h before assaying.
McLaughlin et al.  used fluorescence resonance energy transfer to show that a peptide made of the EGFR JD could sequester PIP2. Using surface plasmon resonance (SPR) as the assay to confirm EGFR/PIP2 interaction, we reproduced these results, showing that a peptide mimicking the EGFR juxtamembrane domain (region 645–662) displayed strong binding to PIP2 (Fig. 1b, top panel). Inspection of the JD of EGFR, as well as other RTKs, shows the presence of a cluster of positively charged residues that alternate with hydrophobic residues (Fig. 1a), The pattern of basic and hydrophobic residues is reminiscent of the basic JDs of most ion channels [8, 13, 21, 35, 52], as well as the PIP2-binding motifs of some cytoskeletal proteins . Neutralization of the first eight positively charged amino acids abolished EGFR/PIP2 interaction in the SPR assay, further highlighting the importance of the eight positively charged amino acids within the 645–662 region for EGFR/PIP2 interaction (Fig. 1b, middle panel). Interestingly, this positively charged region of the EGFR juxtamembrane domain has been shown to be of critical importance for EGFR activation. Deletion of the amino acids between 645 and 657 of the EGFR JD region has been shown to abolish receptor dimerization and autophosphorylation , while a mutant EGFR bearing the same eight mutations as the ones used in our SPR experiments (Fig. 1b, middle panel) has previously been shown to lack tyrosine phosphorylation when heterologously expressed . In view of these earlier reports, our data raise the possibility that EGFR activation and downstream signaling depend on receptor interaction with PIP2. To address this possibility, we first aimed at assessing the role of PIP2-interacting basic residues of the EGFR JD on receptor auto-phosphorylation, using a system appropriate for subsequent physiological studies. X. laevis oocytes provide a heterologous expression system, where ion channel function can be conveniently studied and where the EGFR has been shown to reconstitute with similar properties as in mammalian cells . We used this system to express full length wild-type or mutant EGFR bearing Arg to Asn substitutions within the PIP2 binding domain (see Fig. 2a). An anti total EGFR antibody was used to determine levels of expression of wild-type and mutant EGFR (see Fig. 2b for its specificity), whereas an antibody that specifically recognizes phosphorylated tyrosine 992 of the EGFR (EGFRY992) was used to determine basal and EGF-induced phosphorylation of wild-type and mutant EGFR (Fig. 2c–e). Figure 2c shows that phosphorylation at Y992 of the mutant EGFR bearing the eight Arg to Asn substitutions (ASN8) is clearly impaired, indicating that the same amino acids that are critical for the receptor/PIP2 interaction are also critical for its auto-phosphorylation. From the other two mutants, ASN3 appeared to have the highest impact. Similar results were obtained using a general anti-phosphotyrosine antibody (4 G10; Fig. 2d). Quantification of the data on the EGF-induced EGFR phosphorylation at Y992 from several independent experiments (n=6–8) revealed that the ASN3 mutation was almost as potent as the ASN8 in suppressing EGF-induced Y992 phosphorylation, while mutation of the five distal Arg residues (ASN5) resulted in a weaker, but statistically significant suppression of receptor phosphorylation (Fig. 2e).
To exclude the possibility that the impaired EGFR phosphorylation seen with the ASN3 and ASN8 mutants was due to a decrease in plasma membrane localization, we compared surface expression levels of wild-type EGFR with those of EGFR bearing the ASN3 and ASN8 mutations. Online Resource Figure S1 shows that the mutations did not interfere with the surface expression of the receptor, in good agreement with previous reports showing that neither neutralization , nor deletion  of the 645–657 region of EGFR prevented its expression at the cell surface. Together, our data indicate that neutralization of the basic amino acids between 645 and 657 residues of the EGFR JD region (a) suppressed its autophosphorylation and (b) the three most proximal to the membrane Arg residues (ASN3 or R645–R647) are the most critical for receptor phosphorylation. In agreement with these data, alanine scanning mutagenesis in a recent study by Red Brewer et al.  in 2009 revealed R646 and R647 as the two most critical residues in the entire 645–657 region for EGFR phosphorylation.
To investigate the effect of the above JD mutations on EGF-dependent downstream signaling, we asked whether the mutations affect EGF-dependent regulation of ion channel activity. Xenopus oocytes possess endogenous outwardly rectifying (i.e., conduct more outward than inward current at similar electrochemical driving forces) Ca2+-activated chloride (CaCl) channels that are activated by the Ca2+ released from IP3-dependent Ca2+ stores . Electrophysiological monitoring of CaCl currents (ICaCl) in Xenopus oocytes provides a sensitive assay of intracellular Ca2+ and can therefore serve as a native sensor of Ca2+ release (an alternative to Ca2+-sensitive dyes) that follows stimulation of PLC-coupled receptors (e.g., EGFR, M1 ACh receptors, etc.). Figure 3 shows that oocytes expressing wild-type or mutant EGFR could all elicit at positive potentials outward ICaCl when stimulated by EGF (Fig. 3a, upper traces), thus ensuring that the mutant EGFR could still signal downstream.
We have previously shown that PIP2 hydrolysis through EGFR signaling inhibits the activity of both heteromeric Kir3.1/3.4 and homomeric Kir3.4* (Kir3.4-S143T) inwardly rectifying channels . Inhibition of Kir3 currents provides better quantification of the relative effect of the EGFR mutations on EGF-dependent inhibition than ICaCl, possibly due to the desensitizing character of ICaCl. Thus, we used oocytes co-expressing Kir3.4* together with either wild-type or mutant EGFR. Figure 3b shows that GIRK4* currents prior to inhibition by EGF were at comparable levels of expression regardless of whether they were co-expressed with wild-type or mutant EGFR. Simultaneous monitoring of outwardly and inwardly rectifying currents enabled us to examine whether EGFR signaling took place (outward ICaCl), while at the same time to quantify the extent of the signaling (inhibition of inward Kir3.4* currents). Figure 3a (lower traces) and (summary of data) show that the ASN5 mutant inhibited Kir3.4* currents upon EGF stimulation almost as effectively as the wild-type EGFR. In contrast, the ASN8 and ASN3 mutants, although they were still functional in eliciting ICaCl, were significantly less effective in inhibiting Kir3.4* currents upon EGF stimulation (Fig. 3a, c).
Together, these data show that mutation of the first three Arg residues in the PIP2-binding region of the EGFR basic JD is enough to suppress receptor autophosphorylation and downstream signaling to ion channels, in complete agreement with the alanine scanning mutagenesis data of Red Brewer et al. , reporting R646 and R647 as the two most critical residues in the entire 645–657 region for EGFR phosphorylation. Unlike the above mentioned group, however, we were not able to further narrow down the critical region, as each of the two point mutations that we tested (R645N and R646N) did not result in significant differences in EGF-induced Kir3 current inhibition when compared to wild-type EGFR (data not shown).
To investigate whether PIP2 can modulate EGFR function, we used the PI kinase inhibitor Wortmannin. Generally, Wortmannin inhibits PI(4) kinase (PI4K) at micromolar concentrations, in addition to blocking PI(3) kinase (PI3K), at nanomolar concentrations . PI-4 K is a critical enzyme in the synthetic cascade of PIP2 that catalyzes the addition of phosphate to the 4′ position of the inositol ring of PI, leading to the formation of the PIP2 precursor PI(4)P . Online resource Figure S2 shows that treatment of oocytes with 15 μM of Wortmannin for 1 h reduced PIP2 levels to about 40% of control (see Online resource Figure S2). Thus, in agreement with previously published data , Wortmannin can effectively reduce PIP2 levels. Figure 4a shows a robust, progressive decrease in the EGF-induced EGFRY992 phosphorylation elicited by increasing concentrations of Wortmannin treatment. Wortmannin treatment (100 nM or 15 μM for one hour) reduced EGFRY992 phosphorylation three- and sixfold, respectively (Fig. 4b), whereas the amounts of EGFR present, probed with an anti-total EGFR antibody were not significantly different. A similar inhibition of EGFR phosphorylation was observed when we probed phosphotyrosine at position 1045 (data not shown). Since low concentrations of Wortmannin mainly affect PI-3 K rather than PI4K, the threefold reduction observed with 100 nM of Wortmannin implies a possible contribution of PIP3. This may be specific to the oocyte system, since 100 nM of Wortmannin was ineffective in suppressing EGFR phosphorylation in HeLa cells (see Fig. 5a, b). Even in oocytes however, the additional reduction of EGFR phosphorylation observed upon treatment with 15 μM of Wortmannin (a concentration that reduces PIP2 levels to about 40% of control, see Online Resource Figure S2), suggests that EGFR activation depends on PIP2. Consistent with this hypothesis, earlier work has shown that both PI4K and PIP5K interact with the EGFR .
To further test our hypothesis, we employed synaptojanin, a protein that has also been shown to interact with the EGFR, and whose constitutive PIP2 5-phosphatase activity hydrolyzes PIP2 to PI(4)P . Introduction of synaptojanin mRNA in oocytes decreased EGFRY992 phosphorylation (Fig. 4c, d), further supporting the correlation between decreased PIP2 and inhibition of EGFR phosphorylation. If decreases in PIP2 levels result in diminished EGFR phosphorylation, then an increase in PIP2 levels would be expected to have the opposite effect. Indeed, overexpression of PIP5KIα (a final enzyme in the pathway that leads to PIP2 formation), produced a significant gain in the phosphorylation of wild-type EGFR (Fig. 4e, f). Importantly, phosphorylation of the ASN8 mutant (which does not bind PIP2, see Fig. 1, EGFR645–662MUT) was not affected by PI(5)PKIα overexpression (Fig. 4e, f, see ASN8±PIP5KIα). These findings further support the notion that EGFR activation depends on the levels of PIP2.
To test whether the observed effect of PIP2 on autophosphorylation of heterologously expressed EGFR also applies to mammalian cells with native EGFRs, we used HeLa cells, a cell line of mammalian origin that expresses EGFR endogenously. Figure 5a and b show that EGF-induced phosphorylation of Y992 in the endogenous EGFR of HeLa cells was not significantly affected by 100 nM Wortmannin (overnight application in parallel with serum starvation), but was significantly inhibited by 15-μM Wortmannin.
RNA interference was used to down-regulate the levels of the lipid kinase PIP5KIα, in order to suppress production of PIP2. This treatment indeed led to a significant reduction in the levels of PIP5KIα (Fig. 5c, d), whereas levels of β-tubulin were not affected (see Fig. 5c). Interestingly, HeLa cells transfected with the siRNA for PIP5KIα displayed a marked reduction in both their basal (which was high enough in three out of four batches of cells to allow detection by WB), and EGF-induced EGFRY992 phosphorylation (Fig. 5e and f). The suppression of basal EGFR phosphorylation by PIP5KIα siRNA implies that spontaneous autophosphorylation of the receptor may also be sensitive to PIP2. Alternatively, despite the overnight starvation, depletion of growth factors may not be complete and therefore what is referred to as basal phopshorylation most probably reflects, at least in part, the contribution of residual EGF-induced phosphorylation, which is sensitive to PIP2.
Since it has been reported that it is the PIP5KIβ and not the PIP5KIα isoform that is involved in EGFR internalization, it is unlikely that the siRNA-mediated reduction of the PIP5KIα isoform led to a decrease in EGFR autophosphorylation due to a decreased cell surface receptor density. To explicitly control for such a possibility, we subjected HeLa cells to FACS analysis to determine the cell surface receptor density before and after treatment with siRNA for the PIP5KIα isoform. EGF stimulation indeed caused a reduction in the cell surface receptor density, as has been previously shown . However, treatment with siRNA for the PIP5KIα isoform had no effect on cell surface receptor density before or after EGF treatment (see Online resource Figure S3). Thus, the effect of the siRNA of the PIP5KIα isoform on autophosphorylation of EGFR was not due to changes in receptor internalization.
Phosphoinositides, and in particular phosphatidylinositol (4,5)-bisphosphate, are emerging as central regulators of diverse cellular functions through their effects on transmembrane or plasma membrane associated proteins. Among the best examples of PIP2-sensitive proteins are a long list of ion channels, the activity of which depends on their association with PIP2 [18, 42], and the myristoylated alanine-rich C kinase substrate (MARCKS), which acts as a PIP2 buffer to regulate the availability of PIP2 [10, 30]. Data from the present study place EGFR among the PIP2-dependent/PIP2-associated proteins. Pharmacological or genetic down-regulation of PIP2 levels severely impaired EGFR activation, as measured by receptor auto-phosphorylation, whereas up-regulation of PIP2 potentiated EGF-mediated activation of the receptor.
Our data suggest that specific positively charged residues at the N terminus of the basic juxtamembrane domain of EGFR (645–657) are responsible for receptor interaction with PIP2. Mutation of positively charged residues to neutral ones abolished EGFR/PIP2 interactions and drastically down-regulated receptor activation and signaling to ion channels. Further mutations helped us narrow the critical region to the first three residues of the basic cluster (R645, R646, and R647) as the most critical for receptor activation. In agreement with this conclusion, alanine scanning mutagenesis in a recent study by Red Brewer et al.  revealed R646A and R647A as the two most potent mutations in the entire 645–657 region in terms of suppressing EGFR phosphorylation. With regards to downstream signaling however, although the ASN3 mutation (R645N+R646N+R647N) in our study abolished EGFR-mediated inhibition of Kir3.4* currents, the R645N and R646N single point mutants were not effective.
Several previous reports have highlighted the importance of the EGFR juxtamembrane domain and in particular the 645–657 JM region, on the regulation of receptor activation. Both positive and negative regulatory roles on EGFR activation have been attributed to this region. The inhibitory effects have been attributed to PKC-mediated phosphorylation of Thr-654 at the juxtamembrane region of the EGFR [22, 37], whereas the entire 645–657 region appears to be indispensable for EGFR activation. Deletion of this region has been shown to result in loss of receptor dimerization and trans-autophoshorylation [1, 15, 44], whereas neutralization of the positively charged residue (Arg/Lys to Asn) mutations (the exact same mutations that abolish PIP2 binding, see Fig. 1), severely down-regulated receptor phosphorylation in both NIH3T3 and NR6 cells . Our data suggest that the PIP2-interacting characteristic of the 645–657 region is key to the indispensable role of this region for EGFR activation.
Association of the JM domain of EGFR with PIP2 has previously been shown  and it has been interpreted as a mechanism serving to keep the receptor in an inactive state, when not bound to the ligand . According to the proposed model, upon ligand binding, and receptor dimerization, spontaneous dissociation of a small fraction of the receptors from the membrane could lead to a low level auto-phosphorylation, activation of PLC, IP3-mediated calcium release with a subsequent recruitment of Ca/CaM to the JM domain of the EGFR. This Ca/CaM binding on EGFR would reverse the charge of the juxtamembrane domain, resulting in disengagement of the whole cytoplasmic domain of the receptor from the membrane and exposing the Receptor Tyrosine Kinase domain to phosphorylation and full activation of the receptor. Such a model predicts that prevention of EGFR/PIP2 interaction would favor ligand-independent activation of EGFR and potentiate its EGF-mediated activation . However, as discussed in more detail in , despite this prediction, mutations in the juxtamembrane domain of EGFR that prevent PIP2 binding inactivate the receptor ( and the current study). Since the proposed calmodulin-dependent EGFR regulation requires some level of receptor activation and downstream Ca2+ release , it is reasonable to hypothesize that this may be occurring at a step downstream of the PIP2-dependent regulation that was revealed in the present study.
The precise mechanism of action of PIP2 on the EGFR is unknown. A crystal structure of the EGFR kinase domain by Zhang et al.  shows the formation of an asymmetric dimer between receptor monomers in the activated state. Even though the structure does not include the JD, the authors state that the positioning, flexibility and possible movements of the JD are important, especially since there seems to be a gap between the ends of the dimeric extracellular region (~20 Å apart) and the N-terminal residues of the two kinase monomers (~50 Å apart) . A recent crystal structure by Red Brewer et al. , which includes the JD, confirms the asymmetric dimer between receptor monomers and nearly all of the donor acceptor interactions seen in the previous structure. Similar were the conclusions from another recent study by Jura et al.  who again show that the membrane distal segment of the juxtamembrane region of the receiver/acceptor kinase makes stabilizing contacts with the C-terminal lobe of the activator/donor kinase and propose a model according to which, the membrane-proximal segment of the juxtamembrane region of EGFR (645–663) forms an antiparallel helical dimer that engages the transmembrane helices of the activated receptor to facilitate formation of the asymmetric kinase dimer. The impairment in EGFR activation by the mutations employed in our study most likely reflects both inhibition of PIP2 binding and prevention of asymmetric contact formation. The proposal by Jura et al. activating JD latch resembles the case of erbB2 in which, the transmembrane domains are involved in a rotational sliding of the two monomeric helices against each other to regulate dimerization . Because the JD connects the kinase lobes to the transmembrane segments, such facts point to the JD, which can bind PIP2, as an important regulator of EGFR activation. Thus, the EGFR JD with the aid of PIP2 may have a role similar to the JD segments of the FLT3 receptor tyrosine kinase, i.e., to correctly align and maintain proper register during transition states .
It is well established that receptor-induced PLC activation and PIP2 hydrolysis underlie receptor-mediated inhibition of ion channel activity [16, 17, 52]. Spatiotemporal regulation of PIP2 levels may serve to provide the cell with the appropriate flexibility to fine-tune certain signaling pathways. For example although PIP2 is required for NMDA receptor activity , NMDAR-mediated PLC hydrolysis of PIP2 is necessary for spine remodeling and AMPAR internalization during NMDA receptor-dependent LTD  and mice lacking PLCβ1, the predominant PLC isoform in the forebrain, are LTD deficient . In the case of EGFR, in view of our data showing its dependence on PIP2 for full activation and transmission of EGF-dependent signal, the EGFR-dependent PLC activation and PIP2 hydrolysis may serve as a negative feedback loop to promote receptor inactivation after transmission of the signal to downstream effectors. This auto-inhibitory loop would serve to terminate EGFR-mediated activation of PLC, thus preventing over-depletion of PIP2 due to sustained PLC activity. Consequently, the newly formed PIP2 would be allowed to establish the appropriate environment for a new cycle of EGFR activation.
This work was supported by a National Institutes of Health grant (HL-59949) to DEL. Support for RI and YC was provided by NIH grant DK-38761. We would like to thank Heikki Vaananen, Sophia Gruszecki, Samantha Lee, and Dr. Mei Zhang for excellent technical assistance, Drs. Pietro DeCamilli (Yale University, CT) for the gift of synaptojanin, and Drs. Show-Ling Shyng and Colin Nichols (Oregon Health and Science University, OR, USA and Washington University, MO, USA) for the gift of PI(5)P kinase. We thank Dr. Qi Zhao for help with the analysis of the FACS data. We would also like to thank Drs. Giorgos Panayotou (Fleming Institute, Athens, Greece), Mitchell Goldfarb (Hunter College, NY, USA), Michael Ehlers (Duke Neurobiology, NC, USA), Julia Sable (Columbia University, NY, USA), Tibor Rohacs (UMDNJ), Stuart Aaronson (Department of Oncological Sciences, MSSM) and the Logothetis laboratory members for insightful discussions and comments on the manuscript.
Electronic supplementary material The online version of this article (doi:10.1007/s00424-010-0904-3) contains supplementary material, which is available to authorised users.
Present Address: I. E. Michailidis Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Present Address: R. Rusinova Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10065, USA
Ioannis E. Michailidis, Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY, USA.
Radda Rusinova, Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY, USA.
Anastasios Georgakopoulos, Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, USA.
Yibang Chen, Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY, USA.
Ravi Iyengar, Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY, USA.
Nikolaos K. Robakis, Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, USA.
Diomedes E. Logothetis, Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, 1101 E Marshall St, Richmond, VA 23298, USA.
Lia Baki, Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, 1101 E Marshall St, Richmond, VA 23298, USA.