EGF signalling amplification induced by dynamic clustering of EGFR

Lateral interaction is an important feature of various types of cell surface receptors including the receptor tyrosine kinases (RTKs). Here we report that dynamic lateral interaction produces amplification and variation in signalling of the EGF receptor, a member of RTKs. Binding of EGF is known to induce transphosphorylation inside EGFR dimers. Using single-molecule techniques, the relationship between EGF binding and EGFR phosphorylation has been determined. The number of phosphorylated EGFR molecules became larger than that of EGF binding as unliganded EGFR was phosphorylated, meaning an amplification of EGF signalling. EGFR formed clusters continuously exchanging their elements through thermal diffusion, and direct and/or indirect lateral interactions. As a result, various types of activation sites differing in number of activated receptors were generated. Amplification required no cytoplasmic factors and was observed on semi-intact cells for a wide range of number of EGFR molecules (10(4)-10(6) per cell) suggesting generality of this process.     

妊娠期糖尿病小鼠胎盘EGFR的表达与其发病关系的研究

目的:研究妊娠期糖尿病小鼠胎盘表皮生长因子受体(EGFR)的表达,探讨EGFR表达与妊娠期糖尿病发病的关系。方法:采用链脲佐菌素建立妊娠期糖尿病(gestational diabetes mellitus,GDM)小鼠模型,对照组为正常妊娠小鼠,腹腔注射等量缓冲溶液。测定母鼠体重、血糖;计算胎鼠的存活率;测定胎鼠、胎盘重量,计算胎盘效率;RT-PCR、免疫组化分别测定GDM组和对照组胎盘EGFR m RNA和EGFR蛋白的表达。Pearson相关性分析用于母鼠血糖与EGFR表达的相关性分析。结果:GDM组母鼠体重和血糖均高于对照组(P0.01);GDM组胎鼠、胎盘重量及胎盘效率均高于对照组(P0.01);RT-PCR和免疫组化结果显示GDM组胎盘EGFR m RNA和EGFR蛋白的表达与对照组相比差异有统计学意义(P0.01)。GDM组小鼠血糖值与其胎盘EGFR的表达具有相关性(r=0.582,P0.05)。结论:GDM导致胎盘EGFR表达升高,EGFR并不是GDM的发病因素,EGFR是预防GDM的潜在靶点。     

EGF and angiotensin II modulate lysophosphatidic acid LPA1 receptor function and phosphorylation state

Background

Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA₁ and EGF receptors.

Methods

C9 cells expressing LPA₁ receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA₁ and EGF receptor phosphorylation.

Results

EGF diminished LPA-mediated intracellular calcium response and induced LPA₁ receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA₁ receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA₁ receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events.

Conclusions

EGF receptors and LPA₁ receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA₁ receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA₁ receptor function in inhibitory fashion.

General significance

EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications.     

Oxidation of Analogs of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Monoamine Oxidases A and B and the Inhibition of Monoamine Oxidases by the Oxidation Products

Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+-channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin-2-loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 microM EGTA or 100 microM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 microM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration-dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+-evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.     

Effect of nitrendipine on cardiac and renal lesions and arterial hypertrophy

A low dose of nitrendipine (1 mg/kg twice daily) ameliorated the percent incidence and severity of vascular lesions in the kidney and heart induced by deoxycorticosterone (DOC). Less protection was offered by administration of 1 mg/kg of the calcium antagonist once daily. A lower dose of the antagonist (0.5 mg/kg) administered twice daily produced almost no protection against myocardial scars, but the percent incidence and severity of renal tubular casts and glomerular changes were similar to those following injection of 1 mg/kg of the antagonist twice daily. DOC induced hypertrophy of the media in aorta, coronary artery and renal interlobular artery and renal arteriole. Neither 1 mg/kg once or twice daily nor 0.5 mg twice daily of calcium antagonist modified the hypertrophy of the arterial vasculature in the hypertensive DOC group. We conclude that a low dose of the calcium antagonist dissociates at least in part lesions but not hypertrophy from the increased systolic blood pressure, because the antagonist protects against vascular lesions induced by the hypertension. The antagonist likely acts on the endothelial cell of the vessels alone or combined with an effect on the vascular smooth muscle cells.     

Epidermal growth factor receptors: function modulation by phosphorylation and glycosylation interplay

Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR's functionality.     

Stimulation of tyrosine phosphorylation in lectin treated human lymphocytes

Large increases in tyrosine phosphorylation have been detected in subcellular matrixes isolated from lectin treated human lymphocytes. In lectin stimulated cells proteins of molecular weight 105, 75, 58 and 35 kDa contained phosphotyrosine (P-tyr) whereas non-stimulated cells had no 105 and low levels of P-tyr in proteins of 75, 58 and 35 kDa. In stimulated cells increased tyrosine kinase activity was also shown using gastrin as substrate. In both stimulated and non-stimulated cells the 58 kDa phosphoprotein was the most heavily labelled, after partial proteolysis of the 58 kDa different phosphopeptides were generated. A peptide with a sequence analogous to the autophosphorylated tyrosine site of pp60src inhibited tyrosine phosphorylation in stimulated cells. The lymphocyte system provides a useful tool to study normal tyrosine protein kinases and their role in cellular proliferation.     

Regulation of the phosphorylation of elongation factor 2 by MEK-dependent signalling in adult rat cardiomyocytes

The Gq-coupled agonists phenylephrine and endothelin-1 each activate protein synthesis in cardiomyocytes as part of the programme that leads to cardiac hypertrophy. Here we show that they each induce the dephosphorylation of elongation factor (eEF) 2, a protein that in its dephosphorylated state mediates the translocation step of elongation. The ability of both agonists to induce dephosphorylation of eEF2 requires signalling via the mTOR and MEK/Erk signalling pathways, but is independent of phosphoinositide 3-kinase. Expression of an activated form of MEK leads to dephosphorylation of eEF2, in an mTOR independent manner, indicating that signalling via MEK/Erk suffices to cause dephosphorylation of eEF2.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.     

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

Calcium antagonists cause dry mouth by inhibiting resting saliva secretion

Ca2+ antagonists cause dry mouth by inhibiting saliva secretion. The present study was undertaken to elucidate the mechanism by which Ca2+ antagonists cause dry mouth. Since the intracellular Ca2+ concentration ([Ca2+]i) is closely related to saliva secretion, [Ca2+]i was measured with a video-imaging analysis system by using human submandibular gland (HSG) cells as the material. The Ca2+ antagonist, nifedipine, inhibited the elevation in [Ca2+]i induced by 1-10 microM carbachol (CCh), but had no inhibitory effect on that induced by 30 and 100 microM CCh. The other kinds of Ca2+ antagonists, verapamil (10 microM), diltiazem (10 microM), and the inorganic Ca2+ channel blocker, CdCl2 (50 microM), also inhibited the [Ca2+]i elevation induced by 10 microM CCh. The Ca2+ channel activator, Bay K 8644 (5 microM), significantly enhanced the CCh (10 microM)-induced [Ca2+]i elevation. Endothelin-1 and norepinephrine also increased the CCh (10 microM)-induced [Ca2+]i elevation. SKF-96365 reversed the enhancement of the CCh (10 microM)-induced [Ca2+]i elevation caused by AlF4- and phenylephrine. The phospholipase Cbeta (PLCbeta) inhibitor, U-73122 (5 microM), significantly inhibited the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh, while the PLCbeta activator, m-3M3FBS (20 microM), significantly increased the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh. We therefore conclude that non-selective cation and voltage-dependent Ca2+ channels are involved in resting salivation and that Ca2+ antagonists depress H2O secretion by blocking the Ca2+ channels and thereby cause dry mouth.     

Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II

Cardiac hypertrophy is an important risk factor for heart failure. Epidermal growth factor receptor (EGFR) has been found to play a role in the pathogenesis of various cardiovascular diseases. The aim of this current study was to examine the role of EGFR in angiotensin II (Ang II)‐induced cardiac hypertrophy and identify the underlying molecular mechanisms. In this study, we observed that both Ang II and EGF could increase the phospohorylation of EGFR and protein kinase B (AKT)/extracellular signal‐regulated kinase (ERK), and then induce cell hypertrophy in H9c2 cells. Both pharmacological inhibitors and genetic silencing significantly reduced Ang II‐induced EGFR signalling pathway activation, hypertrophic marker overexpression, and cell hypertrophy. In addition, our results showed that Ang II‐induced EGFR activation is mediated by c‐Src phosphorylation. In vivo, Ang II treatment significantly led to cardiac remodelling including cardiac hypertrophy, disorganization and fibrosis, accompanied by the activation of EGFR signalling pathway in the heart tissues, while all these molecular and pathological alterations were attenuated by the oral administration with EGFR inhibitors. In conclusion, the c‐Src‐dependent EGFR activation may play an important role in Ang II‐induced cardiac hypertrophy, and inhibition of EGFR by specific molecules may be an effective strategy for the treatment of Ang II‐associated cardiac diseases.     

Nuclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654

Nuclear localisation of EGFR is associated with treatment resistance of tumor cells. The aim of this study was to identify molecular targets to block nuclear shuttling of EGFR. Mutation of Thr654, located within the putative EGFR NLS demonstrated that phosphorylation of this residue is essential for nuclear EGFR shuttling following irradiation. Deletion of Thr654 blocked nuclear transport of EGFR, whereas mutation to Glu increased shuttling. Treatment with a peptide, corresponding to the phosphorylated NLS, abolished nuclear EGFR transport and reduced radiation-induced activation of DNA-PK, essential for DNA-repair. In accordance with that, lack of nuclear EGFR increased residual DNA damage in tumor cells and reduced cellular survival following irradiation. Blockage of nuclear EGFR shuttling may be a new strategy to fight treatment resistance.

Structured summary

MINT-7987956: Karyopherin alpha (uniprotkb:P52294) physically interacts (MI:0915) with EGFR (uniprotkb:P00533) by anti bait coimmunoprecipitation (MI:0006)     

Regulation of Nerve Growth Factor Action on Nsp100 Phosphorylation in PC12h Cells by Calcium

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.     

EGF对EGFR基因和c-fos、c-myc原癌基因DNA结合蛋白质的影响

基因转录调控作用是EGF的细胞核内作用之一,而序列特异的DNA结合蛋白质与基因转录的调控密切相关。本文以C_3H小鼠胚胎正常成纤维细胞C_3H_(10)T_(1/2)C18(简称NC_3H_(10))株及其氚标记脱氧胸苷(~3H-TdR)恶性转化的细胞株(简称TC_3H_(10))为对象,我们研究了EGF作用下,核内蛋白质与EGFR基因和c-fos、c-myc二种原癌基因的结合状态。研究发现,EGF的持续作用可使EGFR基因的特异结合蛋白质P120、c-fos基因的特异结合蛋白质P80、以及c-myc基因的特异结合蛋白质P125等明显增加。这些结果提示基因的特异结合蛋白质可能与EGF对基因转录的调控密切相关。上述结果尚未见到国内外同类报道。     

Serrate-Notch signaling defines the scope of the initial denticle field by modulating EGFR activation

The Drosophila embryonic epidermis has been a key model for understanding the establishment of cell type diversity across a cellular field. During segmental patterning, distinct signaling territories are established that employ either the Hedgehog, Spitz, Serrate or Wingless ligands. How these pathways control segmental pattern is not completely clear. One major decision occurs as cells are allocated to differentiate either smooth cuticle or denticle type cuticle. This allocation is based on competition between Wingless signaling and Spitz, which activates the Epidermal Growth Factor Receptor (EGFR). Here we show that a main role for Serrate-Notch signaling is to adjust the Spitz signaling domain. Serrate accomplishes this task by activating Notch in a discrete domain, the main purpose of which is to broaden the spatially regulated expression of Rhomboid. This adjusts the breadth of the source for Spitz, since Rhomboid is necessary for the production of active Spitz. We also show that the Serrate antagonist, fringe, must temper Notch activity to insure that the activation of the EGFR is not too robust. Together, Serrate and Fringe modulate Notch activation to generate the proper level of EGFR activation. If Serrate-Notch signaling is absent, the denticle field narrows while the smooth cell field expands, as judged by the expression of the denticle field determinant Ovo/Shaven baby. This establishes one important role for the Serrate signaling territory, which is to define the extent of denticle field specification.     

大鼠和小鼠睾丸表皮生长因子表达的免疫组织化学定位观察

为了了解大鼠和小鼠睾丸是否产生ＥＧＦ及其细胞定位,本实验用ＥＧＦ单克隆抗体对大鼠和小鼠睾丸进行了免疫细胞化学定位研究,结果显示：（１）出生后,大鼠和小鼠睾丸即开始产生ＥＧＦ,分泌活动主要位于睾丸间质细胞。（２）至性成熟期,少数精原细胞、精母细胞及个别圆形精子细胞和管周肌样细胞也产生ＥＧＦ,使生精小管尤其是血睾屏障管腔小室侧的ＥＧＦ分泌增加。（３）在本实验中,睾丸支持细胞未见明显ＥＧＦ阳性染色。结果表明,大鼠和小鼠睾丸是可以产生ＥＧＦ的,间质细胞是其主要的ＥＧＦ分泌细胞。进入性成熟期后,少数精原细胞、精母细胞及个别圆形精子细胞和管周肌样细胞也产生ＥＧＦ。大鼠和小鼠睾丸在发育过程中ＥＧＦ分泌量呈上升趋势,至性成熟期达分泌高峰