Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.     

The Constitutive Activity of Epidermal Growth Factor Receptor vIII Leads to Activation and Differential Trafficking of Wild-type Epidermal Growth Factor Receptor and erbB2

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII''s activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII''s activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. (J Histochem Cytochem 58:529–541, 2010)     

Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.Both constitutive endocytosis and activated endocytosis are highly regulated events by which cells take up nutrients and internalize receptors for recycling or degradation (47). Endocytosed molecules are delivered to early endosomes, where the components are sorted to the cell surface for recycling back to the plasma membrane, or to late endosomes to be degraded in lysosomes (17). The molecular mechanisms regulating these events are not fully understood.One of the major protein families involved in the trafficking of membrane compartments is sorting nexins (SNXs), which are characterized by the presence of phox homology (PX) domains (8, 65). The PX domain is a protein module which consists of approximately 130 amino acids with three β-strands followed by three α-helices forming a helical subdomain, and the general function of this module is to interact with the head groups of inositol phospholipids through which parental proteins are targeted to specific cellular compartments. Most of the SNXs examined to date specifically recognize phosphatidylinositol 3-phosphate [PtdIns(3)P], which is found predominately in early endosomes (11). The founding member of the SNX family, SNX1, was initially identified as an interaction partner of epidermal growth factor receptor (EGFR), and the expression of SNX1 enhanced lysosomal degradation of EGFR (38); therefore, SNXs are most likely to be involved in the trafficking of many different families of receptors which are recycled to the cell surface or sent to the lysosome for degradation (19). On the other hand, PX domain-containing proteins have also been reported to bind to phosphoinositides other than PtdIns(3)P and to have functions independent of receptor trafficking (54). For example, phospholipase D is a PX domain-containing protein that hydrolyzes phosphatidylcholine to produce a second-messenger molecule, phosphatidic acid. Interestingly, phospholipase D has been recently shown to accelerate EGFR endocytosis by activating dynamin GTPase through its PX domain but independently of lipase activity (39). Cytokine-independent survival kinase (CISK) is a PX domain-containing protein kinase that has also been shown to regulate sorting of a chemokine receptor CXCR4 through AIP4, the CXCR4 ubiquitin ligase (60). RGS-PX1, a GTPase-activating protein for Gαs of heterotrimeric GTP-binding proteins, and KIF16B, a PX domain-containing kinesin superfamily member, have been shown to regulate EGFR trafficking (27, 72) and are now grouped into the SNX family as SNX13 and SNX26, respectively.Another feature of the PX domain is a well-conserved polyproline sequence (PXXP) in the variable loop between α1 and α2 helices, which led to the original identification of the PX domain as a SH3 domain-binding partner (53). The physiological importance of both intermolecular and intramolecular interactions mediated by polyproline sequences has been shown in various molecules, including phospholipase D2 (33) and p47^phox (1). In mammals, there are currently 47 proteins harboring PX domains, and 30 proteins are termed SNXs (59). The functions of these proteins have just begun to be revealed.Actin cytoskeletal dynamics have been implicated not only in cell motility and cytokinesis but also in endocytic processes, although the necessity and role in endocytosis in higher eukaryotic cells remain ambiguous (12, 34, 35, 55). The WASP homology 2 (WH2) domain is known as an actin-binding motif found in regulators of the actin cytoskeleton, including Wiskott-Aldrich syndrome protein (WASP), Scar/WASP-family verprolin-homologous protein (WAVE), verprolin/WASP-interacting protein (WIP), missing in metastasis (MIM), and β-thymosins (52). Some proteins with WH2 domains, such as β-thymosin, prevent actin filament assembly by sequestering actin monomers, while others, such as N-WASP and the Drosophila protein Ciboulot participate in barbed-end actin assembly (52). Recently, the structural basis for these opposite functions of WH2 domains was demonstrated; the interaction of the C-terminal region of β-thymosin/WH2 domain with the pointed end of the actin monomer accounts for the switch in function from inhibition to promotion of actin assembly (26). WH2 domains exist in almost 20 proteins, whose functions remain to be clarified.In the present study, we isolated a new multimodular protein (termed PXK), conserved in multicellular organisms including humans through flies, which possesses a PX domain, a protein kinase-like domain, and a WH2 domain. We show that the PX and WH2 domains function as PtdIns(3)P and actin-binding domains, respectively. PXK expression in COS cells accelerated ligand-induced EGFR endocytosis and degradation that was dependent on a functional PX domain but independent of the WH2 domain. PXK also enhanced ubiquitination of EGFR induced by EGF stimulation in these cells. Based on these results, we propose that PXK is a functional sorting nexin that may play an additional role in cellular function via its interaction with the actin cytoskeleton.     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Microenvironmental Stiffness Enhances Glioma Cell Proliferation by Stimulating Epidermal Growth Factor Receptor Signaling

The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling.     

表皮生长因子及受体在甲状腺肿瘤中的表达

目的:研究表皮生长因子(Epidermal Growth Factor,EGF)及受体(Epidermal Growth Factor Receptor,EGFR)及在甲状腺肿瘤中的表达。方法:应用免疫组织化学法检测91例甲状腺病变组织中EGFR和EGF的表达情况。结果:结节性甲状腺肿、甲状腺腺瘤、分化型甲状腺癌标本中EGFR表达的阳性率分别为15%、25%、68.62%,EGF表达的阳性率分别为10%、15%、68.62%,其中EGFR、EGF在分化型甲状腺癌与其余两组间差异均有统计学意义(P<0.05)。EGFR和EGF在甲状腺乳头状癌中的表达与性别、年龄、肿瘤大小、淋巴结转移、临床分期等临床因素无明显相关。结论:EGF和EGFR的表达可作为鉴别甲状腺肿瘤良恶性的一个指标。     

Intracellular Transactivation of the Insulin-like Growth Factor I Receptor by an Epidermal Growth Factor Receptor

Growth factor receptors may be transactivated not only by homologous receptors, but also by heterologous receptors. We have investigated this possibility, using for this purpose R⁻/EGFR cells, which are mouse embryo cells devoid of IGF-I receptors, but overexpressing the EGF receptor. At variance with mouse embryo cells with a wild-type number of IGF-I receptors and overexpressing the EGF receptor, R⁻/EGFR cells cannot grow in EGF only, nor can they form colonies in soft agar. However, if a wild type human IGF-I receptor is stably transfected into R⁻/EGFR cells, growth in EGF and colony formation in soft agar are restored. To determine a possible interaction between the two receptors, we transfected into R⁻/EGFR cells a number of IGF-I receptor mutants with different impaired functions. The only IGF-I receptor that cannot reverse the growth phenotype of R⁻/EGFR cells is a receptor with a point mutation at the ATP-binding site. All other mutant receptors, even when incapable of responding to IGF-I with a mitogenic signal, made R⁻/EGFR cells fully capable of responding with growth to EGF stimulation. IGF-I receptor mutants that are mitogenic but not transforming made R⁻/EGFR cells grow in EGF only, but were incapable of inducing the transformed phenotype. The mutant IGF-I receptors are activated (tyrosyl phosphorylation of IRS-1) in response to EGF. These experiments indicate that certain IGF-I receptor mutants with loss of function can be reactivated intracellularly by an overexpressed EGF receptor and confirm that the C-terminus of the IGF-IR is required for its transforming activity.     

50 Hz弱磁场诱导离体表皮生长因子受体聚集

利用原子力显微镜(atomic force microscope,AFM)观测到浓度为5滋g/mL离体表皮因子受体(epidermal growth factor receptor,EGFR)蛋白在50Hz磁场作用下发生聚集,其颗粒呈现倍数变大和变高趋势;利用透射电镜(transmission electronic microscope,TEM)也得到类似的蛋白变大趋势。结果表明,在AFM和TEM下观察到,磁场作用后蛋白聚集颗粒的平均半高宽(平均直径)由(21.7±2.2)nm(12.5nm)增大到(33.0±4.0)nm(23nm),最可几高度由(1.42±0.18)nm增加到(3.08±0.17)nm。这种由磁场引起的聚集效应呈时间依赖性。利用Alexa-488-EGF标记EGFR观察了磁场暴露对细胞上EGF受体表达的影响,提示EGF受体的表达可能稍有上调。上述结果提示50Hz磁场信号可能通过影响EGFR的膜上聚集状态来影响下游信号通路。这种对信号通路的影响可能是电磁场生物性效应的一种机制。     

HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

Regulation of Proliferation and Apoptosis by Epidermal Growth Factor and Protein Kinase C in Human Ovarian Surface Epithelial Cells

Epidermal growth factor (EGF) is produced in the ovary and influences proliferation of the malignant ovarian surface epithelium (OSE); yet its role in malignancy or in regulating the normal surface epithelium is unclear. In human OSE cells derived from primary cultures of normal tissue transfected with SV40 large T antigen (IOSE cells), EGF promoted survival but not proliferation. This survival effect was reversed by acute treatment with the phorbol ester, 12-0-tetradecanoyl-13-phorbol acetate (TPA) which alone markedly inhibited IOSE proliferation. We tested whether the activities of the mitogen-activated protein kinases (ERK1/2 and JNK1) varied in response to EGF, TPA, or combinations of these agonists and if the same treatments altered patterns of immediate early gene expression. Alone, EGF activated ERK1/2, increased and sustained levels of c-junmRNA, but had almost no effect on JNK1 activation. Conversely, PKC activation resulted in a rapid, but transient induction of c-fosRNA and of both kinases, JNK1 and ERK2. When combined, EGF and TPA further enhanced the phosphorylation of both enzymes despite inhibiting survival. Though JNKs and ERKs are thought to transduce opposing cellular responses, in IOSE cells, robust costimulation of the JNK and ERK pathways may redirect the survival message.     

Regulation of Growth Inhibitory Factor Expression by Epidermal Growth Factor and Interleukin-1β in Cultured Rat Astrocytes

Growth inhibitory factor (GIF) is highly expressed in the CNS under physiological conditions, but its expression is reduced in neurodegenerative diseases, such as Alzheimer's disease. The results of this study show that the levels of GIF and GIF mRNA were not influenced by neuroglial interactions. GIF was highly expressed in confluent astrocytes, but the expression was down-regulated in low-density growing astrocytes. A high level of GIF was not observed in serum-starved low-density cultures. These findings suggest that GIF is a quiescent state-specific protein and that two different mechanisms may exist for the cells to enter the quiescent state. Among interleukin-1beta (IL-1beta), fibroblast growth factor-2, epidermal growth factor (EGF), amyloid beta1-42, and 50% O2, only EGF and IL-1beta altered the level of GIF in confluent astrocytes: EGF increased both GIF mRNA and protein, and IL-1beta decreased GIF mRNA, but did not alter GIF protein. Kinetic analysis of the GIF mRNA level revealed the biphasic regulation of GIF mRNA expression by IL-1beta, i.e., a transient up-regulation followed subsequently by down-regulation, explaining in part the discrepancy between the levels of GIF mRNA and protein in astrocytes treated with IL-1beta.     

Regulation of Epidermal Growth Factor Receptor Signaling and Erlotinib Sensitivity in Head and Neck Cancer Cells by miR-7

Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3′-untranslated region (3′-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 target genes, providing insight into the tumor suppressor function of miR-7. Furthermore, we identified several target miR-7 mRNAs with a putative role in the sensitization of FaDu cells to erlotinib. Together, these data support the coordinate regulation of Akt signaling by miR-7 in HNC cells and suggest the therapeutic potential of miR-7 alone or in combination with EGFR TKIs in this disease.     

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.     

Epidermal Growth Factor Induces Proliferation and DNA Synthesis in Ciliate Cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis Gl. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G ₁ and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells in a stationary culture (G ₀ phase) whose development is independent of the growth factors in the medium.     

Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.     

Novel Mechanisms of Fibroblast Growth Factor Receptor 1 Regulation by Extracellular Matrix Protein Anosmin-1

Activation of fibroblast growth factor (FGF) signaling is initiated by a multiprotein complex formation between FGF, FGF receptor (FGFR), and heparan sulfate proteoglycan on the cell membrane. Cross-talk with other factors could affect this complex assembly and modulate the biological response of cells to FGF. We have previously demonstrated that anosmin-1, a glycosylated extracellular matrix protein, interacts with the FGFR1 signaling complex and enhances its activity in an IIIc isoform-specific and HS-dependent manner. The molecular mechanism of anosmin-1 action on FGFR1 signaling, however, remains unknown. Here, we show that anosmin-1 directly binds to FGFR1 with high affinity. This interaction involves domains in the N terminus of anosmin-1 (cysteine-rich region, whey acidic protein-like domain and the first fibronectin type III domain) and the D2–D3 extracellular domains of FGFR1. In contrast, anosmin-1 binds to FGFR2IIIc with much lower affinity and displays negligible binding to FGFR3IIIc. We also show that FGFR1-bound anosmin-1, although capable of binding to FGF2 alone, cannot bind to a FGF2·heparin complex, thus preventing FGFR1·FGF2·heparin complex formation. By contrast, heparin-bound anosmin-1 binds to pre-formed FGF2·FGFR1 complex, generating an anosmin-1·FGFR1·FGF2·heparin complex. Furthermore, a functional interaction between anosmin-1 and the FGFR1 signaling complex is demonstrated by immunofluorescence co-localization and Transwell migration assays where anosmin-1 was shown to induce opposing effects during chemotaxis of human neuronal cells. Our study provides molecular and cellular evidence for a modulatory action of anosmin-1 on FGFR1 signaling, whereby binding of anosmin-1 to FGFR1 and heparin can play a dual role in assembly and activity of the ternary FGFR1·FGF2·heparin complex.FGF⁵ signaling plays an important role in a wide range of fundamental biological responses (1–3). Both FGF and FGFR bind to heparan sulfate (HS) and heparin, a highly sulfated type of HS produced in connective tissue mast cells. Heparan sulfate proteoglycans (HSPG) are the cell surface co-receptors essential for the formation of functional FGF·FGFR signaling complex (4, 5). There are four structurally related FGFRs (FGFR1–4), which consist of an extracellular ligand-binding region containing three immunoglobulin (Ig)-like domains (D1–D3), a single transmembrane domain, and a cytoplasmic domain with protein-tyrosine kinase catalytic activity. The 22 members of the FGF family bind to the interface formed by the D2/D3 domains and the linker between these domains (6, 7), whereas a conserved positively charged region in D2 serves as the HS binding site (8). An unusual stretch of seven to eight acidic residues designated as the “acid box” is present in the linker connecting D1 and D2. Alternative splicing events occur to generate various isoforms, including a truncated receptor lacking D1 and the D1–D2 linker or a full-length receptor that differs in the second half of D3, designated as IIIb and IIIc isoforms (5). Two crystal structures have been proposed to demonstrate how the FGF·FGFR·heparin complex is assembled (9, 10). Recent evidence suggests that both may be biologically relevant (11, 12).The diversity of FGF signaling pathways and consequent biological functions require that activation of FGFR should be tightly regulated. Such regulation can occur either at the level of the extracellular receptor-ligand complex assembly or via intracellular modulation of downstream effectors (13). Extracellular regulation mainly involves the interaction between each component of the FGF·FGFR·HS signaling complex. For example, FGF8 is shown to bind mostly to the FGFR IIIc isoforms, whereas FGF7 acts as the preferential ligand for the FGFR2 IIIb isoform (13, 14). Sequence specificity, length, and sulfation patterns of HS are also important regulators of the FGF·FGFR interaction (15, 16).Cell surface proteins other than FGFs and HSPGs participate in FGFR signaling regulation. FLRT3 (a member of the fibronectin-leucine-rich transmembrane protein family) promotes FGF signaling and interacts with FGFR1 and FGFR4 via its extracellular fibronectin type III (FnIII) domain (17). Sef (similar expression to fgf genes) functions as an antagonist of FGF signaling in zebrafish. The two FnIII regions of Sef are essential for its function and interaction with FGFR1 and FGFR2 (18). Neuronal cell adhesion molecule (NCAM), N-cadherin, and L1 have also been identified as functionally relevant in FGFR-mediated neurite outgrowth (19–22). The FnIII domains of NCAM bind to the D2 and D3 domains of FGFR1 (19) and FGFR2 (23) to induce ligand-independent receptor phosphorylation.Anosmin-1, an extracellular matrix-associated glycosylated protein, appears to be a novel member of the extracellular FGFR signaling modulators (24, 25). Loss-of-function mutations of anosmin-1 and FGFR1 are associated with Kallmann syndrome (KS), underlying X-linked, and autosomal dominant/recessive inheritance mode, respectively (26–28). KS is a human developmental genetic disorder characterized by loss of sense of smell (anosmia) caused by abnormal olfactory bulb development and delayed, even arrested puberty caused by disrupted migration of the gonadotropin-releasing hormone (GnRH)-secreting neuron. We previously reported that anosmin-1 acts as an FGFR1IIIc isoform-specific co-ligand, which enhances signaling activity. In human embryonic GnRH olfactory neuroblast FNC-B4 cells, anosmin-1 induced neurite outgrowth and cytoskeletal rearrangements through FGFR1-dependent mechanisms involving p42/44 and p38 mitogen-activated protein kinases and Cdc42/Rac1 activation (25). A functional interaction is also demonstrable between anosmin-1 and FGFR1 in optic nerve oligodendrocyte precursor development (24). Structurally, anosmin-1 comprises an N-terminal cysteine-rich domain (CR) and a whey acidic protein-like (WAP) domain, followed by four tandem FnIII repeats and a C-terminal histidine rich region (Fig. 1a). Current evidence suggests that anosmin-1 functions by affecting FGF2-induced activation of FGFR1 signaling rather than by directly stimulating the receptor. However, the precise molecular mechanism of this interaction remains unclear.Open in a separate windowFIGURE 1.Generation of recombinant anosmin-1, anosmin-1 mutants, FGFR1D1D3, and FGFR1D2D3 proteins. a, the schematic structures of recombinant proteins of anosmin-1 and FGFR1. Each domain in the wild type (PIWF4), point mutants (mPIWF4^N267K, mPIWF4^E514K, and mPIWF4^F517L), and truncated (PIWF1, PIWF2, and PIF4) anosmin-1 protein analogues are represented by a shaded rectangle. V5 and 6His epitopes at the C terminus are represented by a clear rectangle. Each immunoglobulin-like domain in the full ectodomain (FGFR1D1D3) and truncated form (FGFR1D2D3) of FGFR1 is represented by a half circle. The acid box (AB) is represented by a filled rectangle. H, histidine-rich region. b, 0.5–1 μg of purified recombinant proteins are loaded in each lane and visualized by colloidal blue staining. Molecular mass markers in kilodaltons are shown on the left.We now report for the first time that anosmin-1 directly binds to FGFR1 using surface plasmon resonance (SPR), chemical cross-linking, and immunofluorescence co-localization studies in living cells. This interaction occurs between the N-terminal CR, WAP, and the first FnIII domain of anosmin-1 and D2 and D3 ectodomains of FGFR1. Moreover, SPR studies using sequential injections and Transwell migration assays in immortalized FNC-B4-hTERT cells suggest that anosmin-1 can have opposing effects in the formation and activation of the FGF2·FGFR1·heparin complex depending on the order of their binding interactions with anosmin-1.     

Bile Acid-induced Epidermal Growth Factor Receptor Activation in Quiescent Rat Hepatic Stellate Cells Can Trigger Both Proliferation and Apoptosis

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47^phox and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Cζ, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H₂O₂), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.Hydrophobic bile acids play a major role in the pathogenesis of cholestatic liver disease and are potent inducers of hepatocyte apoptosis by triggering a ligand-independent activation of the CD95² death receptor (1–5). The underlying molecular mechanisms are complex and involve a Yes-dependent, but ligand-independent activation of the epidermal growth factor receptor (EGFR), which catalyzes CD95-tyrosine phosphorylation as a prerequisite for CD95 oligomerization, formation of the death-inducing signaling complex (DISC), and apoptosis induction (6, 7). Bile acids also activate EGFR in cholangiocytes (8) and activated hepatic stellate cells (HSC) (9), however, the mechanisms underlying bile acid-induced EGFR activation in HSC remained unclear (9). Surprisingly, bile acid-induced EGFR activation in HSC does not trigger apoptosis but results in a stimulation of cell proliferation (9). The behavior of quiescent HSC toward CD95 ligand (CD95L) is also unusual. CD95L, which is a potent inducer of hepatocyte apoptosis (10–12), triggers activation of the EGFR in quiescent HSC, stimulates HSC proliferation, and simultaneously inhibits CD95-dependent death signaling through CD95-tyrosine nitration (13). Similar observations were made with other death receptor ligands, i.e. tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) (13). The mitogenic action of CD95L in quiescent, 1–2-day cultured HSC is because of a c-Src-dependent shedding of EGF and subsequent auto/paracrine activation of the EGFR (13). This unusual behavior of quiescent HSC toward death receptor ligands may relate to the recent findings that quiescent HSC might represent a stem/progenitor cell compartment in the liver with a capacity to differentiate not only into myofibroblasts but also toward hepatocyte- and endothelial-like cells (14). Thus, stimulation of HSC proliferation and resistance toward apoptosis in the hostile cytokine milieu accompanying liver injury may help HSC to play their role in liver regeneration. During cholestatic liver injury quiescent HSC are exposed to increased concentrations of circulating bile acids, but it is not known whether this may lead to HSC proliferation (as shown for activated HSC) (9), HSC apoptosis (as shown for hepatocytes) (1–7), or both of them. Therefore, the aim of the current study was (a) to identify the molecular mechanisms underlying bile acid-induced EGFR activation and (b) to elucidate whether bile acid-induced signaling can couple to both cell proliferation and cell death in quiescent HSC.The present study shows that cholestatic bile acids trigger a rapid NADPH oxidase activation in quiescent HSC, which leads to a Yes-mediated EGFR phosphorylation and HSC proliferation. In contrast to hepatocytes, hydrophobic bile acids do not induce a JNK signal in HSC. However, when JNK activation is induced by coadministration of either cycloheximide (CHX) or hydrogen peroxide (H₂O₂), the bile acid-induced mitogenic signal is shifted to an apoptotic one.     

Pgrmc1 (Progesterone Receptor Membrane Component 1) Associates with Epidermal Growth Factor Receptor and Regulates Erlotinib Sensitivity

Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and metabolism. Epidermal growth factor receptor (EGFR) is a transmembrane receptor-tyrosine kinase that is associated with cancer progression, and the EGFR inhibitors erlotinib/tarceva and tyrphostin/AG-1478 are potent anti-cancer therapeutics. Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b₅-related protein that is up-regulated in tumors and promotes cancer growth. Pgrmc1 and its homologues have been implicated in cell signaling, and we show here that Pgrmc1 increases susceptibility to AG-1478 and erlotinib, increases plasma membrane EGFR levels, and co-precipitates with EGFR. Pgrmc1 co-localizes with EGFR in cytoplasmic vesicles and co-fractionates with EGFR in high density microsomes. The findings have therapeutic potential because a Pgrmc1 small molecule ligand, which inhibits growth in a variety of cancer cell types, de-stabilized EGFR in multiple tumor cell lines. EGFR is one of the most potent receptor-tyrosine kinases driving tumorigenesis, and our data support a role for Pgrmc1 in promoting several cancer phenotypes at least in part by binding EGFR and stabilizing plasma membrane pools of the receptor.