小脑颗粒神经元去极化诱导的上皮生长因子受体间接激活信号传导途径是由谷氨酸受体所介导
|
摘要
前言
细胞外信号调节激酶1和2(ERK_(1/2))磷酸化苏氨酸/丝氨酸残基形成活化形式的ERK_(1/2)(p-ERK_(1/2))。该磷酸化及其下游的信号传导通路在神经元发育过程中具有重要意义。在成熟脑内,ERK_(1/2)的磷酸化与记忆形成和突触可塑性密切相关。早期研究表明,将培养的神经元、PC12细胞以及脑片暴露在高钾、谷氨酸或谷氨酸受体激动剂条件下,可引起p-ERK_(1/2)水平升高。
上皮生长因子受体(EGFR)间接激活是一种由外界刺激或G蛋白耦联受体(GPCR)激活,导致某种生长因子的释放,进而激活自身和相邻的酪氨酸激酶受体,产生与直接激活酪氨酸激酶(RTK)相似效应的过程。它是近年来所发现一种新的信号传导途径。EGFR间接激活对于调节神经元本身功能及其周围细胞的功能具有重要作用。
在EGFR间接激活过程中,G蛋白耦联受体的激活和去极化引起的细胞内游离钙离子浓度升高,均可导致金属蛋白酶催化的EGFR配体的释放,进而活化自身以及相邻细胞的上皮生长因子受体。在EGFR配体家族中,转化生长因子-α(TGF-α)、肝素结合上皮生长因子(HB-EGF)在体内小脑颗粒神经元和小脑外颗粒层上表达。有关其它配体的表达,目前尚不明确。
原代培养7-8天小脑颗粒神经元,谷氨酸能受体分化成熟,其中包括N-甲基-D-天冬氨酸受体(NMDAR)、α-氨基-3-羟基-5-甲基-4-异恶唑受体(AMPAR)和代谢型谷氨酸受体(mGluR)。本篇文章主要研究高钾去极化引起原代培养小脑颗粒神经元ERK_(1/2)磷酸化的机制。即(1)上皮生长因子受体间接激活是否参与高钾去极化引起的小脑颗粒神经元ERK_(1/2)磷酸化。(2)高钾去极化引起小脑颗粒神经元ERK_(1/2)磷酸化过程中,是否依赖于谷氨酸受体的激活。(3)EGFR相关配体在原代培养的小脑颗粒神经元上的表达。(4)PKC是否参与上皮生长因子受体间接激活信号传导途径。
方法
采用原代培养的小脑颗粒神经元。将细胞在3℃无血清相关培养液中孵育5-60分钟,含有5mM、10mM、25mM、45mM[K~+]_e或50μM谷氨酸以及相关特异性抑制剂。应用冰PBS洗涤细胞,加入裂解液,终止反应,收集细胞。进行凝胶电泳,免疫印迹分析。ERK_(1/2)在本文中仅用做样品加样的监测指标,而在统计分析中未涉及ERK_(1/2)。使用SPSS12.0软件进行统计学分析,多组资料用one-wayANOVA方法进行比较,P＜0.05表示差异具有统计学意义。
结果
1、高钾诱导ERK_(1/2)磷酸化
高钾在小脑颗粒神经元引起p-ERK_(1/2)升高。在[K~+]_e浓度为10mM、25mM时,磷酸化水平略有升高,在25mM时,p-ERK_1组升高具有统计学意义。在[K~+]_e浓度为45mM时,磷酸化水平明显升高,且具有统计学意义。[K~+]_e引起ERK_(1/2)磷酸化的时间相对较晚,45mM[K~+]_e作用在10分钟后,p-ERK_(1/2)开始升高,20分钟后升高具有统计学意义,在40分钟时下降,60分钟后再次升高具有统计学意义。
2、上皮生长因子家族相关成员mRNA表达
在原代培养8天的小脑颗粒神经元及成年小鼠小脑内,HB-EGF、TGF-α均有表达。但EGF(上皮生长因子)和Amphiregulin(双向调节蛋白)在神经元和小脑内均无表达。
3、TRK、金属蛋白酶抑制剂和肝素的作用
应用上皮生长因子受体抑制剂1μM AG1478可以抑制45mM[K~+]_e20分钟引起的ERK_(1/2)磷酸化,差异具有统计学意义。而AG1478对45mM[K~+]_e60分钟引起ERK_(1/2)磷酸化不完全抑制,差异具有统计学意义。
10μM Zn~(2+)依赖性金属蛋白酶抑制剂GM6001,能够阻断45mM[K~+]_e20分钟引起的ERK_(1/2)磷酸化。1mg/ml肝素能够抑制此磷酸化反应,差异具有统计学意义。
4、谷氨酸和其受体拮抗剂的作用
应用50μM谷氨酸作用于神经元,可引起与去极化相似的ERK_(1/2)磷酸化。50μM谷氨酸引起小脑颗粒神经元ERK_(1/2)磷酸化,此反应可以被AG1478、GM6001所抑制。NMDA受体拮抗剂MK801(1μM)和非NMDA受体拮抗剂CNQX(10μM)也能够抑制谷氨酸引起的ERK_(1/2)磷酸化。另外,NMDA受体拮抗剂MK801和非NMDA受体拮抗剂CNQX也可抑制45mM[K~+]_e在20分钟和60分钟引起的ERK_(1/2)磷酸化。
5、在不含谷氨酰胺的培养液中细胞外高钾的作用
将神经元在不含谷氨酸前体-谷氨酰胺的盐溶液中孵育,细胞外的谷氨酸浓度明显降低,高钾诱导的谷氨酸释放也被抑制。将细胞暴露45mM[K~+]_e20分钟和60分钟可引起ERK_(1/2)的磷酸化升高,但AG1478无抑制作用。表明在此条件下,高钾刺激的ERK_(1/2)的磷酸化不依赖于EGFR间接激活。
6、PKC抑制剂的影响
将神经元在不含谷氨酰胺培养液中孵育,45mM[K~+]_e在20分钟时引起的ERK_(1/2)的磷酸化,PKC抑制剂GF109203X不能抑制该磷酸化反应。在含有谷氨酰胺的培养液中,高钾引起的ERK_(1/2)的磷酸化升高主要是由于去极化导致谷氨酸释放增加。此反应在20分钟时可被GF109203X抑制。但是对60分钟时同样条件下ERK_(1/2)的磷酸化没有影响。在含有谷氨酰胺的培养液中,50μM的谷氨酸刺激5分钟后ERK_(1/2)的磷酸化增加可被PKC的抑制剂不完全抑制。虽然50μM的谷氨酸条件下PKC抑制剂抑制作用轻微,但是明显超过了没有谷氨酸条件下PKC抑制剂的作用。
讨论
目前研究表明,上皮生长因子家族成员至少有7种:HB-EGF、TGF-α、EGF、Amphiregulin、BTC(β-细胞素)、EPR(表皮调节素)和epigen。仅前四种在脑内表达,其中HB-EGF、TGF-α存在于小脑及原代培养颗粒神经元。应用原位杂交技术检测在体内小脑颗粒神经元上有TGF-α的表达。HB-EGF在发育初期脑内表达水平达到高峰,并在脑内持续表达。EGF在前脑和皮层星形胶质细胞存在表达,而在原代培养的小脑颗粒神经元以及成年小脑内均无表达。
上皮生长因子受体家族(ErbB家族)广泛存在于脑内,包括ErbB1(EGFR)、ErbB2、ErbB3和ErbB4,并且已证实在原代培养的小脑颗粒神经元上表达。该受体激活引起受体酪氨酸激酶磷酸化,进而激活有丝分裂活化蛋白激酶(包括ERK_(1/2))和磷脂酰肌醇3激酶(PI3K)。ErbB4对于神经元迁移和突触可塑性具有重要意义。小脑颗粒神经元中表达的神经体调节蛋白是一种ErbB4的激动剂,能够诱导放射胶质细胞形成和颗粒细胞的迁移。在原代培养小脑颗粒神经元中,EGF能够引起MAP激酶活化。
基质金属蛋白酶MMP2、3、9在小脑内表达,可能参与间接激活过程。在对皮质扩散性抑制的研究中发现,伴随着大量K~+和谷氨酸的释放,位于大脑皮层的MMP9发生活化。
在神经元上皮生长因子受体间接激活的研究中,早期发现该途径存在于GT1-7细胞系(促性腺激素释放激素下丘脑神经元细胞系)中。另外,经去极化、缓激肽以及cAMP处理的PC12细胞(嗜铬细胞瘤细胞系)也存在上皮生长因子受体间接激活信号传导通路。然而,目前在原代培养的神经元中尚无EGFR间接激活报道。该信号传导途径所涉及的生长因子释放相当复杂,而且可能具有细胞特异性、G蛋白耦联受体特异性和发育生物学特异性。最近我们实验室发现,在原代培养星形胶质细胞中,α-肾上腺素能受体激动剂右旋美托咪啶通过G蛋白βγ亚基,蛋白激酶C、Scr激酶、金属蛋白酶以及TRK活化,从而引起ERK_(1/2)的磷酸化。在原代培养的小脑颗粒神经元中,星形胶质细胞约占总含量的5-10%,主要表达代谢性谷氨酸受体,目前尚无NMDA受体在星形胶质细胞表达的报道。所以,星形胶质细胞上皮生长因子受体的激活不可能成为去极化引起小脑颗粒神经元ERK_(1/2)的磷酸化的机制。
本课题研究表明在原代培养的小脑颗粒神经元由高钾去极化诱导的ERK_(1/2)的磷酸化是一个相当复杂的过程。高钾去极化条件下在20和60分钟引起谷氨酸释放进而引起ERK_(1/2)磷酸化。仅在20分钟时ERK_(1/2)磷酸化依赖于PKC活性。在相同条件下,50μM的谷氨酸5分钟引起的ERK_(1/2)的磷酸化增加仅部分依赖于PKC活性,说明其对PKC抑制剂不敏感。但高钾去极化在20和60分钟时所诱发ERK_(1/2)磷酸化并不完全依赖于EGFR的间接激活,并且20分钟时也不依赖于PKC的活化。在原代培养的小脑颗粒神经元中,NMDA受体抑制剂MK801和非NMDA受体抑制剂CNQX可以阻断高钾去极化及谷氨酸引起的ERK_(1/2)磷酸化,提示该过程依赖于NMDA受体和非NMDA受体的激活,这与Baron等人在海马切片上发现一致,CNQX可以完全阻断KCI引起ERK_(1/2)磷酸化,而NMDA受体拮抗剂AP5也部分抑制该反应。在对小脑颗粒神经元研究中,早期发现NMDA受体的激活可以引起ERK_(1/2)磷酸化,但以前的研究均没有阐述是否存在上皮生长因子受体间接激活。
最近研究发现,小脑的颗粒细胞突触上存在谷氨酸NMDA受体和PKC依赖性的LTP。在对小脑切片有关的神经递质释放与钙离子动力学的研究表明,谷氨酸受体激活后引起大量Ca~(2+)进入细胞内,使电压依从Ca~(2+)通道开放,进一步释放游离Ca~(2+),从而使细胞游离Ca~(2+)增加。另外,小脑的颗粒细胞的激活并不依赖突触的刺激,而主要通过树突电压依从性的钙通道开放,使细胞游离Ca~(2+)增加引起的。上述发现提示,谷氨酸受体介导和去极化诱导的ERK_(1/2)磷酸化在小脑颗粒神经元的重要性,但有关PKC在其中的具体作用目前还不很清楚。
结论
原代培养7-8天小鼠小脑颗粒神经元,高钾引起ERK_(1/2)磷酸化,该过程依赖于谷氨酸的释放和谷氨酸NMDA和非NMDA受体调节。高钾去极化引起小脑颗粒神经元谷氨酸的释放激活金属蛋白酶,活化的金属蛋白酶水解HB-EGF(可能包括TGF-α)的脱落,进而激活EGFR,导致ERK_(1/2)磷酸化。
Introduction
Phosphorylation of extracellular-signal regulated kinase (ERK) 1 and 2 (ERK_(1/2)) generates extracellular-signal regulated kinase 1 and extracellular-signal regulated kinase 2 phosphorylated at serine/threonine residues (p-ERK_(1/2)), the active form of ERK_(1/2). This activation and ensuing downstream signaling is important for many neuronal functions during development and in the adult brain, where it plays a major role in plasticity and thus in memory formation. It is consistent with these functions that phosphorylation of ERK_(1/2) occurs both in the intact nervous system in response to afferent stimulation and during exposure of cultured neurons, PC12 cells or brain slices to a depolarizing concentration of potassium ions (K~+), to the excitatory neurotransmitter glutamate, or to subtype-specific glutamate agonists.
Epiithelial growth factor receptor (EGFR) transactivation in which signaling to a G-protein-coupled receptor (GPCR) leads to release ('shedding') of a growth factor, which in turn stimulates receptor tyrosine-kinase (RTK) on the same of adjacent cells and evoked by directly obtained by direct RTK stimulation. It is a novel signaling pathway which plays a majoy role in neuron survival and function.
In the transactivation process of the EGFR, activation of G_(i/o) protein-coupled receptors or a depolarization- or transmitter-mediated increase in free cytosolic calcium concentration ([Ca~(2+)]_i) leads to metalloproteinase-catalyzed shedding of an EGFR agonist, which stimulates EGFRs on the same cell or its neighbor(s). Among EGFR agonists transforming growth factor-α(TGF-α) and heparin-binding epidermal growth factor (HB-EGF) are expressed in cerebellar granule cells and the cerebellar external granule layer, but there is no information whether other EGFR agonists are present.
7-8-day-old primary cultures of cerebellar granule neurons constitute a well-established, well-differentiated glutamatergic preparation, expressing NMDA, AMPA and metabotropic glutamate receptors. In the present study we have studied the mechanism of ERK_(1/2) phosphorylation during K~+-mediated depolarization using 7-8-day-old primary cultures of cerebellar granule neurons. (1) K~+-mediated depolarization is transactivation of epidermal growth factor (EGF) receptors (EGFRs). (2) K~+-induced depolarization is mediated by glutmate receptor. (3) EGFR family expression in the primary cultures of cerebellar granule neuron.
Methods
The primary cerebellar granule neuron culture were the pre-incubate in the corresponding medium without serum at 37℃at a [K~+]_e of 5, 10, 25 or 45 mM in the absence or presence of specific inhibitors. The reaction was stopped by washing with ice-cold phosphate-buffered saline (PBS) containing 7.5 mM glucose, and the cells were scraped off the dishes and harvested in 0.5 ml of ice-cold buffer for Western-Blot. Concentration of glutamate released were measured by high-pressure liquid chromatography. The results are analysised with one-way ANOVA by SPSS12.0 software. P<0.05 indicate the statically significant difference.
Results
1. [K~+]_e induced ERK_(1/2) phosphorylation
Exposure to raised [K~+]_e for 20 min caused a concentration-dependent increase of p-ERK_(1/2), whereas there was no significant change in total content of ERK_(1/2) (results not presented). A small response of ERK_(1/2) appeared to occur at an [K~+]_e concentration of 10 mM and was larger at 25 mM and a large and statistically significant increase was seen at 45 mM [K~+]_e. The stimulation of ERK_(1/2) activities by high [K+]e occurred relatively slowly. After 10 min of exposure to 45 mM [K~+]_e, p-ERK_1 and p-ERK_2 increased slightly and reached a statistically significant maximum after 20 min, followed by a decrease to basal values at 40 min and a second, similar increase at 60 min.
2. mRNA expression of EGF-related family members
The response to a depolarizing [K~+]_e might be due to a transactivation of the EGFR, since mRNAs of both HB-EGF and TGF-αwere expressed in 8-day-old cerebellar granule cells in primary cultures at similar levels as in cerebellum of adult mice . In contrast, neither the cultured cells nor cerebellum of adult mice expressed EGF or amphiregulm mRNA. Betacellulin, epiregulin and epigen were not tested for, since their presence has not been reported in brain.
3. Effects of inhibitors of TRK and metalloproteinase and of heparin
The effect of AG 1478, a specific EGFR tyrosine kinase inhibitor, was tested in order to establish if transactivation was involved. In the presence of 1μM AG 1478 phosphorylation of ERK_1 and ERK_2 by 45 mM [K~+]_e for 20 min was inhibited. The inhibitory effects on phosphorylation of ERK_1 and ERK_2 were not significantly different from each other and they were statistically significant in both cases. After exposure to 45 mM [K~+]_e for 60 min, the response was indistinguishable from that seen after 20 min of exposure although the inhibition may have been less complete. Additional inhibitors of the transactivation process were therefore only tested after 20 min of K~+ exposure.Ten micromolar GM 6001, an inhibitor of Zn~(2+)-dependent metalloproteinase, affected the response to 20 min of exposure to 45 mM [K~+]_e in exactly the same manner as AG 1478. In the presence of 1 mg/ml of heparin, an inhibitor of HB-EGF and amphiregulin, there was also a significant inhibition of the response to 45 mM [K~+]_e, but the inhibition was less complete than in the case of the two other inhibitors. This was reflected by a statistically significant difference between the presence of the inhibitor alone and in the additional presence of elevated [K~+]_e.
4. Effects of glutamate and antagonists of NMDA and of non-NMDA receptors
To establish if the transactivation-dependent ERK_(1/2) phosphorylation resulted from K~+-mediated depolarization as such or a glutamate release resulting from the depolarization, experiments were carried out in which 50 uM glutamate was added instead of elevating [K~+]_e. Exposure to glutamate for 5 min caused a significant increase in ERK_(1/2) phosphorylation, which was abolished by AG 1478 and GM 6001. The effect of glutamate was also inhibited by both 1μM of MK-801, an antagonist of NMDA glutamate receptors, and 10μM of CNQX, an antagonist of non-NMDA ionotropic glutamate receptors . One micromolar MK-801 also inhibited the stimulation by 45 mM [K~+]_e, regardless whether the [K~+]_e exposure lasted 20 or 60 min, although an apparent difference between the control value and the value in the presence of elevated [K~+]_e plus MK-801 after 60 min of exposure suggested that the inhibition was not complete. Ten micromolar CNQX also inhibited K~+-mediated ERK phosphorylation.
5. Effects of elevated [K~+]_e in glutamine-free medium
In the absence of extracellular glutamine the extracellular glutamate concentration was significantly lowered (from 1.8 to 1.1μM at 5 mM [K~+]_e), and the K~+-induced glutamate release was inhibited (a non-significant increase to 9.3 uM in the glutamine-deprived medium versus a significant increase to 36 uM in the presence of glutamine). Nevertheless, also in this situation exposure to 45 mM [K~+]_e stimulated ERK_(1/2) phosphorylation at both 20 and 60 min, but AG 1478 had no effect, indicating that although K~+-mediated depolarization as such was able to induce ERK_(1/2) phosphorylation, this phosphorylation was not dependent upon transactivation.
6. Effects of PKC inhibition
GF109203X, an inhibitor of PKC, had no effect on ERK_(1/2) phosphorylation under resting condition (5 mM [K~+]_e). It did also not affect the stimulation by 45 mM [K~+]_e after 20 min . However, the effect of 50μM glutamate was inhibited. This inhibition was not complete, at least not in the case of ERK_2, where phosphorylation in the combined presence of 50μM [K~+]_e and the inhibitor slightly, but significantly, exceeded that in the presence of GF 109203X without glutamate.
Consistent with the sensitivity to GF109203X of the glutamate-mediated increase of ERK_(1/2) phosphorylation after 20 min, K~+-mediated stimulation of ERK_(1/2) phosphorylation in glutamine-replete medium, known mainly to reflect the effect of glutamate released as a result of the depolarization was also inhibited by GF109203X. However, the similar ERK_(1/2) phosphorylation after 60 min was unaffected by the presence of the PKC inhibitor.
Discussion
Seven different members of the EGF growth factor superfamily have been identified. Only the first four have been demonstrated in brain tissue. Among these HB-EGF and TGF-αwere found to be present in cerebellum and in cultured cerebellar granule neurons. This is consistent with a previous observation that TGF-αis expressed in cerebellar granule cells in the brain in vivo, as shown by in situ hybridization. Also, HB-EGF is expressed at high level in brain in vivo at early developmental stages; it declines thereafter, although some HB-EGF remains in the cerebellum. In contrast, EGF and amphiregulin were undetectable both in cerebellar granule cells in primary cultures and cerebellum from adult mouse, consistent with previous findings.
Transactivation of neuronal EGFRs has previously been reported in GT1-7 cells, immortalized hypothalamic neurons, treated with gonadotropin-releasing hormone. It has also been found in PC12 cells, a phaeochromocytoma cell line, during depolarization or exposure to bradykinin or cAMP. However, to our knowledge transactivation of EGFRs has not previously been reported in primary cultures of neurons. The signal pathway inducing "shedding" of growth factors is complex and may be cell type-, G-protein coupled receptor-, and developmental stage-specific.
A significant portion of K~+-mediated ERK_(1/2) phosphorylation depended upon NMDA receptor activation, as indicated by its inhibition by NMDA, an antagonist of the NMDA receptor. ERK_(1/2) phosphorylation in cerebellar granule cells in response to NMDA receptor stimulation has previously been demonstrated by Sato et al. and Zhu et al., but these authors did not investigate whether transactivation was involved.
In the present cultures the initial K~+-mediated glutamate release amounts to about 10 nmol/min per mg protein, but due to a time-dependent reduction of release and to concentration-dependent reuptake the glutamate concentration in the medium is only 10-15 nmol/mg protein after 30 min of incubation. With approximately 1 mg cell protein incubated in 2 ml of medium, this amount corresponds to a medium glutamate concentration of 5-8 uM, and perhaps more in the synaptic clefts. This concentration is likely to suffice for activation of NMDA receptors, which have a high affinity for glutamate, but perhaps not for AMPA receptors which have a much lower affinity. This may explain why CNQX, an antagonist at AMPA receptors, had no effect, whereas Wu et al. and Limatola et al., using addition of receptor agonists have demonstrated ERK_(1/2) phosphorylation induced by AMPA receptor activation in cerebellar granule cells. Again, it was not investigated whether transactivation was involved.
The present report together with our previous paper show a remarkable complexity of the signaling pathways between K~+-mediated depolarization of cerebellar granule cells in primary cultures and ERK_(1/2) phosphorylation . They include a glutamate-induced ERK_(1/2) phosphorylation secondary to K~+-evoked glutamate release at both 20 and 60 min, which is dependent upon EGFR transactivation. The present study has shown that only the former of these two increases in ERK_(1/2) phosphorylation requires PKC activity. The PKC dependence of the response to glutamate after 20 min was confirmed by addition of 50 uM glutamate to glutamine-free medium, although a minor part of this effect appeared to be PKC-independent. In addition the K+-mediated depolarization per se causes ERK_(1/2) phosphorylation at the same time points. However, this phosphorylation occurs independently of transactivation and is also independent of PKC activation, at least at 20 min. PKC inhibition was not tested in glutamine-free medium at 60 min but there was no indication of any PKC-dependent component at 60 min in the glutamine-replete medium.
Accumulating evidence suggests that ERK phosphorylation may play an important role in at least certain types of long-term potentiation (LTP) and learning. Recently, NMDA- and PKC-dependent LTP has been demonstrated at the cerebellar mossy fiber-granule cell synapse. Also, investigation of cytosolic [Ca~(2+)] dynamics during repetitive neurotransmission in cerebellar slices showed that glutamate receptor-mediated [Ca~(2+)]j increase in granule cell dendrites was locally amplified by opening of voltage-dependent Ca~(2+) channels and Ca~(2+) release from intracellular stores. Moreover, granule cell firing contributed to elevating cytosolic [Ca~(2+)] through voltage-dependent channels also in the dendrites that were not synaptically stimulated. These findings attest to the importance of both glutamate-mediated and depolarization-mediated ERK phosphorylation in cerebellar granule cells but it is not known where PKC activity fits into the picture.
Conclusion
In conclusion, depolarization of 7-8-day old cerebellar granule neurons, obtained from 7-day-old mice, by exposure to 45 mM extracellular [K~+]_e leads to phosphorylation of ERK_(1/2), mediated by NMDA receptor activation and partly by a transactivation process, leading to release of HB-EGF and perhaps also of TGF-α. The subsequent action of these EGFR agonists may play a major role for the effects of NMDA receptor activation on brain plasticity.
细胞外信号调节激酶1和2(ERK_(1/2))磷酸化苏氨酸/丝氨酸残基形成活化形式的ERK_(1/2)(p-ERK_(1/2))。该磷酸化及其下游的信号传导通路在神经元发育过程中具有重要意义。在成熟脑内,ERK_(1/2)的磷酸化与记忆形成和突触可塑性密切相关。早期研究表明,将培养的神经元、PC12细胞以及脑片暴露在高钾、谷氨酸或谷氨酸受体激动剂条件下,可引起p-ERK_(1/2)水平升高。
上皮生长因子受体(EGFR)间接激活是一种由外界刺激或G蛋白耦联受体(GPCR)激活,导致某种生长因子的释放,进而激活自身和相邻的酪氨酸激酶受体,产生与直接激活酪氨酸激酶(RTK)相似效应的过程。它是近年来所发现一种新的信号传导途径。EGFR间接激活对于调节神经元本身功能及其周围细胞的功能具有重要作用。
在EGFR间接激活过程中,G蛋白耦联受体的激活和去极化引起的细胞内游离钙离子浓度升高,均可导致金属蛋白酶催化的EGFR配体的释放,进而活化自身以及相邻细胞的上皮生长因子受体。在EGFR配体家族中,转化生长因子-α(TGF-α)、肝素结合上皮生长因子(HB-EGF)在体内小脑颗粒神经元和小脑外颗粒层上表达。有关其它配体的表达,目前尚不明确。
原代培养7-8天小脑颗粒神经元,谷氨酸能受体分化成熟,其中包括N-甲基-D-天冬氨酸受体(NMDAR)、α-氨基-3-羟基-5-甲基-4-异恶唑受体(AMPAR)和代谢型谷氨酸受体(mGluR)。本篇文章主要研究高钾去极化引起原代培养小脑颗粒神经元ERK_(1/2)磷酸化的机制。即(1)上皮生长因子受体间接激活是否参与高钾去极化引起的小脑颗粒神经元ERK_(1/2)磷酸化。(2)高钾去极化引起小脑颗粒神经元ERK_(1/2)磷酸化过程中,是否依赖于谷氨酸受体的激活。(3)EGFR相关配体在原代培养的小脑颗粒神经元上的表达。(4)PKC是否参与上皮生长因子受体间接激活信号传导途径。
方法
采用原代培养的小脑颗粒神经元。将细胞在3℃无血清相关培养液中孵育5-60分钟,含有5mM、10mM、25mM、45mM[K~+]_e或50μM谷氨酸以及相关特异性抑制剂。应用冰PBS洗涤细胞,加入裂解液,终止反应,收集细胞。进行凝胶电泳,免疫印迹分析。ERK_(1/2)在本文中仅用做样品加样的监测指标,而在统计分析中未涉及ERK_(1/2)。使用SPSS12.0软件进行统计学分析,多组资料用one-wayANOVA方法进行比较,P＜0.05表示差异具有统计学意义。
结果
1、高钾诱导ERK_(1/2)磷酸化
高钾在小脑颗粒神经元引起p-ERK_(1/2)升高。在[K~+]_e浓度为10mM、25mM时,磷酸化水平略有升高,在25mM时,p-ERK_1组升高具有统计学意义。在[K~+]_e浓度为45mM时,磷酸化水平明显升高,且具有统计学意义。[K~+]_e引起ERK_(1/2)磷酸化的时间相对较晚,45mM[K~+]_e作用在10分钟后,p-ERK_(1/2)开始升高,20分钟后升高具有统计学意义,在40分钟时下降,60分钟后再次升高具有统计学意义。
2、上皮生长因子家族相关成员mRNA表达
在原代培养8天的小脑颗粒神经元及成年小鼠小脑内,HB-EGF、TGF-α均有表达。但EGF(上皮生长因子)和Amphiregulin(双向调节蛋白)在神经元和小脑内均无表达。
3、TRK、金属蛋白酶抑制剂和肝素的作用
应用上皮生长因子受体抑制剂1μM AG1478可以抑制45mM[K~+]_e20分钟引起的ERK_(1/2)磷酸化,差异具有统计学意义。而AG1478对45mM[K~+]_e60分钟引起ERK_(1/2)磷酸化不完全抑制,差异具有统计学意义。
10μM Zn~(2+)依赖性金属蛋白酶抑制剂GM6001,能够阻断45mM[K~+]_e20分钟引起的ERK_(1/2)磷酸化。1mg/ml肝素能够抑制此磷酸化反应,差异具有统计学意义。
4、谷氨酸和其受体拮抗剂的作用
应用50μM谷氨酸作用于神经元,可引起与去极化相似的ERK_(1/2)磷酸化。50μM谷氨酸引起小脑颗粒神经元ERK_(1/2)磷酸化,此反应可以被AG1478、GM6001所抑制。NMDA受体拮抗剂MK801(1μM)和非NMDA受体拮抗剂CNQX(10μM)也能够抑制谷氨酸引起的ERK_(1/2)磷酸化。另外,NMDA受体拮抗剂MK801和非NMDA受体拮抗剂CNQX也可抑制45mM[K~+]_e在20分钟和60分钟引起的ERK_(1/2)磷酸化。
5、在不含谷氨酰胺的培养液中细胞外高钾的作用
将神经元在不含谷氨酸前体-谷氨酰胺的盐溶液中孵育,细胞外的谷氨酸浓度明显降低,高钾诱导的谷氨酸释放也被抑制。将细胞暴露45mM[K~+]_e20分钟和60分钟可引起ERK_(1/2)的磷酸化升高,但AG1478无抑制作用。表明在此条件下,高钾刺激的ERK_(1/2)的磷酸化不依赖于EGFR间接激活。
6、PKC抑制剂的影响
将神经元在不含谷氨酰胺培养液中孵育,45mM[K~+]_e在20分钟时引起的ERK_(1/2)的磷酸化,PKC抑制剂GF109203X不能抑制该磷酸化反应。在含有谷氨酰胺的培养液中,高钾引起的ERK_(1/2)的磷酸化升高主要是由于去极化导致谷氨酸释放增加。此反应在20分钟时可被GF109203X抑制。但是对60分钟时同样条件下ERK_(1/2)的磷酸化没有影响。在含有谷氨酰胺的培养液中,50μM的谷氨酸刺激5分钟后ERK_(1/2)的磷酸化增加可被PKC的抑制剂不完全抑制。虽然50μM的谷氨酸条件下PKC抑制剂抑制作用轻微,但是明显超过了没有谷氨酸条件下PKC抑制剂的作用。
讨论
目前研究表明,上皮生长因子家族成员至少有7种:HB-EGF、TGF-α、EGF、Amphiregulin、BTC(β-细胞素)、EPR(表皮调节素)和epigen。仅前四种在脑内表达,其中HB-EGF、TGF-α存在于小脑及原代培养颗粒神经元。应用原位杂交技术检测在体内小脑颗粒神经元上有TGF-α的表达。HB-EGF在发育初期脑内表达水平达到高峰,并在脑内持续表达。EGF在前脑和皮层星形胶质细胞存在表达,而在原代培养的小脑颗粒神经元以及成年小脑内均无表达。
上皮生长因子受体家族(ErbB家族)广泛存在于脑内,包括ErbB1(EGFR)、ErbB2、ErbB3和ErbB4,并且已证实在原代培养的小脑颗粒神经元上表达。该受体激活引起受体酪氨酸激酶磷酸化,进而激活有丝分裂活化蛋白激酶(包括ERK_(1/2))和磷脂酰肌醇3激酶(PI3K)。ErbB4对于神经元迁移和突触可塑性具有重要意义。小脑颗粒神经元中表达的神经体调节蛋白是一种ErbB4的激动剂,能够诱导放射胶质细胞形成和颗粒细胞的迁移。在原代培养小脑颗粒神经元中,EGF能够引起MAP激酶活化。
基质金属蛋白酶MMP2、3、9在小脑内表达,可能参与间接激活过程。在对皮质扩散性抑制的研究中发现,伴随着大量K~+和谷氨酸的释放,位于大脑皮层的MMP9发生活化。
在神经元上皮生长因子受体间接激活的研究中,早期发现该途径存在于GT1-7细胞系(促性腺激素释放激素下丘脑神经元细胞系)中。另外,经去极化、缓激肽以及cAMP处理的PC12细胞(嗜铬细胞瘤细胞系)也存在上皮生长因子受体间接激活信号传导通路。然而,目前在原代培养的神经元中尚无EGFR间接激活报道。该信号传导途径所涉及的生长因子释放相当复杂,而且可能具有细胞特异性、G蛋白耦联受体特异性和发育生物学特异性。最近我们实验室发现,在原代培养星形胶质细胞中,α-肾上腺素能受体激动剂右旋美托咪啶通过G蛋白βγ亚基,蛋白激酶C、Scr激酶、金属蛋白酶以及TRK活化,从而引起ERK_(1/2)的磷酸化。在原代培养的小脑颗粒神经元中,星形胶质细胞约占总含量的5-10%,主要表达代谢性谷氨酸受体,目前尚无NMDA受体在星形胶质细胞表达的报道。所以,星形胶质细胞上皮生长因子受体的激活不可能成为去极化引起小脑颗粒神经元ERK_(1/2)的磷酸化的机制。
本课题研究表明在原代培养的小脑颗粒神经元由高钾去极化诱导的ERK_(1/2)的磷酸化是一个相当复杂的过程。高钾去极化条件下在20和60分钟引起谷氨酸释放进而引起ERK_(1/2)磷酸化。仅在20分钟时ERK_(1/2)磷酸化依赖于PKC活性。在相同条件下,50μM的谷氨酸5分钟引起的ERK_(1/2)的磷酸化增加仅部分依赖于PKC活性,说明其对PKC抑制剂不敏感。但高钾去极化在20和60分钟时所诱发ERK_(1/2)磷酸化并不完全依赖于EGFR的间接激活,并且20分钟时也不依赖于PKC的活化。在原代培养的小脑颗粒神经元中,NMDA受体抑制剂MK801和非NMDA受体抑制剂CNQX可以阻断高钾去极化及谷氨酸引起的ERK_(1/2)磷酸化,提示该过程依赖于NMDA受体和非NMDA受体的激活,这与Baron等人在海马切片上发现一致,CNQX可以完全阻断KCI引起ERK_(1/2)磷酸化,而NMDA受体拮抗剂AP5也部分抑制该反应。在对小脑颗粒神经元研究中,早期发现NMDA受体的激活可以引起ERK_(1/2)磷酸化,但以前的研究均没有阐述是否存在上皮生长因子受体间接激活。
最近研究发现,小脑的颗粒细胞突触上存在谷氨酸NMDA受体和PKC依赖性的LTP。在对小脑切片有关的神经递质释放与钙离子动力学的研究表明,谷氨酸受体激活后引起大量Ca~(2+)进入细胞内,使电压依从Ca~(2+)通道开放,进一步释放游离Ca~(2+),从而使细胞游离Ca~(2+)增加。另外,小脑的颗粒细胞的激活并不依赖突触的刺激,而主要通过树突电压依从性的钙通道开放,使细胞游离Ca~(2+)增加引起的。上述发现提示,谷氨酸受体介导和去极化诱导的ERK_(1/2)磷酸化在小脑颗粒神经元的重要性,但有关PKC在其中的具体作用目前还不很清楚。
结论
原代培养7-8天小鼠小脑颗粒神经元,高钾引起ERK_(1/2)磷酸化,该过程依赖于谷氨酸的释放和谷氨酸NMDA和非NMDA受体调节。高钾去极化引起小脑颗粒神经元谷氨酸的释放激活金属蛋白酶,活化的金属蛋白酶水解HB-EGF(可能包括TGF-α)的脱落,进而激活EGFR,导致ERK_(1/2)磷酸化。
Introduction
Phosphorylation of extracellular-signal regulated kinase (ERK) 1 and 2 (ERK_(1/2)) generates extracellular-signal regulated kinase 1 and extracellular-signal regulated kinase 2 phosphorylated at serine/threonine residues (p-ERK_(1/2)), the active form of ERK_(1/2). This activation and ensuing downstream signaling is important for many neuronal functions during development and in the adult brain, where it plays a major role in plasticity and thus in memory formation. It is consistent with these functions that phosphorylation of ERK_(1/2) occurs both in the intact nervous system in response to afferent stimulation and during exposure of cultured neurons, PC12 cells or brain slices to a depolarizing concentration of potassium ions (K~+), to the excitatory neurotransmitter glutamate, or to subtype-specific glutamate agonists.
Epiithelial growth factor receptor (EGFR) transactivation in which signaling to a G-protein-coupled receptor (GPCR) leads to release ('shedding') of a growth factor, which in turn stimulates receptor tyrosine-kinase (RTK) on the same of adjacent cells and evoked by directly obtained by direct RTK stimulation. It is a novel signaling pathway which plays a majoy role in neuron survival and function.
In the transactivation process of the EGFR, activation of G_(i/o) protein-coupled receptors or a depolarization- or transmitter-mediated increase in free cytosolic calcium concentration ([Ca~(2+)]_i) leads to metalloproteinase-catalyzed shedding of an EGFR agonist, which stimulates EGFRs on the same cell or its neighbor(s). Among EGFR agonists transforming growth factor-α(TGF-α) and heparin-binding epidermal growth factor (HB-EGF) are expressed in cerebellar granule cells and the cerebellar external granule layer, but there is no information whether other EGFR agonists are present.
7-8-day-old primary cultures of cerebellar granule neurons constitute a well-established, well-differentiated glutamatergic preparation, expressing NMDA, AMPA and metabotropic glutamate receptors. In the present study we have studied the mechanism of ERK_(1/2) phosphorylation during K~+-mediated depolarization using 7-8-day-old primary cultures of cerebellar granule neurons. (1) K~+-mediated depolarization is transactivation of epidermal growth factor (EGF) receptors (EGFRs). (2) K~+-induced depolarization is mediated by glutmate receptor. (3) EGFR family expression in the primary cultures of cerebellar granule neuron.
Methods
The primary cerebellar granule neuron culture were the pre-incubate in the corresponding medium without serum at 37℃at a [K~+]_e of 5, 10, 25 or 45 mM in the absence or presence of specific inhibitors. The reaction was stopped by washing with ice-cold phosphate-buffered saline (PBS) containing 7.5 mM glucose, and the cells were scraped off the dishes and harvested in 0.5 ml of ice-cold buffer for Western-Blot. Concentration of glutamate released were measured by high-pressure liquid chromatography. The results are analysised with one-way ANOVA by SPSS12.0 software. P<0.05 indicate the statically significant difference.
Results
1. [K~+]_e induced ERK_(1/2) phosphorylation
Exposure to raised [K~+]_e for 20 min caused a concentration-dependent increase of p-ERK_(1/2), whereas there was no significant change in total content of ERK_(1/2) (results not presented). A small response of ERK_(1/2) appeared to occur at an [K~+]_e concentration of 10 mM and was larger at 25 mM and a large and statistically significant increase was seen at 45 mM [K~+]_e. The stimulation of ERK_(1/2) activities by high [K+]e occurred relatively slowly. After 10 min of exposure to 45 mM [K~+]_e, p-ERK_1 and p-ERK_2 increased slightly and reached a statistically significant maximum after 20 min, followed by a decrease to basal values at 40 min and a second, similar increase at 60 min.
2. mRNA expression of EGF-related family members
The response to a depolarizing [K~+]_e might be due to a transactivation of the EGFR, since mRNAs of both HB-EGF and TGF-αwere expressed in 8-day-old cerebellar granule cells in primary cultures at similar levels as in cerebellum of adult mice . In contrast, neither the cultured cells nor cerebellum of adult mice expressed EGF or amphiregulm mRNA. Betacellulin, epiregulin and epigen were not tested for, since their presence has not been reported in brain.
3. Effects of inhibitors of TRK and metalloproteinase and of heparin
The effect of AG 1478, a specific EGFR tyrosine kinase inhibitor, was tested in order to establish if transactivation was involved. In the presence of 1μM AG 1478 phosphorylation of ERK_1 and ERK_2 by 45 mM [K~+]_e for 20 min was inhibited. The inhibitory effects on phosphorylation of ERK_1 and ERK_2 were not significantly different from each other and they were statistically significant in both cases. After exposure to 45 mM [K~+]_e for 60 min, the response was indistinguishable from that seen after 20 min of exposure although the inhibition may have been less complete. Additional inhibitors of the transactivation process were therefore only tested after 20 min of K~+ exposure.Ten micromolar GM 6001, an inhibitor of Zn~(2+)-dependent metalloproteinase, affected the response to 20 min of exposure to 45 mM [K~+]_e in exactly the same manner as AG 1478. In the presence of 1 mg/ml of heparin, an inhibitor of HB-EGF and amphiregulin, there was also a significant inhibition of the response to 45 mM [K~+]_e, but the inhibition was less complete than in the case of the two other inhibitors. This was reflected by a statistically significant difference between the presence of the inhibitor alone and in the additional presence of elevated [K~+]_e.
4. Effects of glutamate and antagonists of NMDA and of non-NMDA receptors
To establish if the transactivation-dependent ERK_(1/2) phosphorylation resulted from K~+-mediated depolarization as such or a glutamate release resulting from the depolarization, experiments were carried out in which 50 uM glutamate was added instead of elevating [K~+]_e. Exposure to glutamate for 5 min caused a significant increase in ERK_(1/2) phosphorylation, which was abolished by AG 1478 and GM 6001. The effect of glutamate was also inhibited by both 1μM of MK-801, an antagonist of NMDA glutamate receptors, and 10μM of CNQX, an antagonist of non-NMDA ionotropic glutamate receptors . One micromolar MK-801 also inhibited the stimulation by 45 mM [K~+]_e, regardless whether the [K~+]_e exposure lasted 20 or 60 min, although an apparent difference between the control value and the value in the presence of elevated [K~+]_e plus MK-801 after 60 min of exposure suggested that the inhibition was not complete. Ten micromolar CNQX also inhibited K~+-mediated ERK phosphorylation.
5. Effects of elevated [K~+]_e in glutamine-free medium
In the absence of extracellular glutamine the extracellular glutamate concentration was significantly lowered (from 1.8 to 1.1μM at 5 mM [K~+]_e), and the K~+-induced glutamate release was inhibited (a non-significant increase to 9.3 uM in the glutamine-deprived medium versus a significant increase to 36 uM in the presence of glutamine). Nevertheless, also in this situation exposure to 45 mM [K~+]_e stimulated ERK_(1/2) phosphorylation at both 20 and 60 min, but AG 1478 had no effect, indicating that although K~+-mediated depolarization as such was able to induce ERK_(1/2) phosphorylation, this phosphorylation was not dependent upon transactivation.
6. Effects of PKC inhibition
GF109203X, an inhibitor of PKC, had no effect on ERK_(1/2) phosphorylation under resting condition (5 mM [K~+]_e). It did also not affect the stimulation by 45 mM [K~+]_e after 20 min . However, the effect of 50μM glutamate was inhibited. This inhibition was not complete, at least not in the case of ERK_2, where phosphorylation in the combined presence of 50μM [K~+]_e and the inhibitor slightly, but significantly, exceeded that in the presence of GF 109203X without glutamate.
Consistent with the sensitivity to GF109203X of the glutamate-mediated increase of ERK_(1/2) phosphorylation after 20 min, K~+-mediated stimulation of ERK_(1/2) phosphorylation in glutamine-replete medium, known mainly to reflect the effect of glutamate released as a result of the depolarization was also inhibited by GF109203X. However, the similar ERK_(1/2) phosphorylation after 60 min was unaffected by the presence of the PKC inhibitor.
Discussion
Seven different members of the EGF growth factor superfamily have been identified. Only the first four have been demonstrated in brain tissue. Among these HB-EGF and TGF-αwere found to be present in cerebellum and in cultured cerebellar granule neurons. This is consistent with a previous observation that TGF-αis expressed in cerebellar granule cells in the brain in vivo, as shown by in situ hybridization. Also, HB-EGF is expressed at high level in brain in vivo at early developmental stages; it declines thereafter, although some HB-EGF remains in the cerebellum. In contrast, EGF and amphiregulin were undetectable both in cerebellar granule cells in primary cultures and cerebellum from adult mouse, consistent with previous findings.
Transactivation of neuronal EGFRs has previously been reported in GT1-7 cells, immortalized hypothalamic neurons, treated with gonadotropin-releasing hormone. It has also been found in PC12 cells, a phaeochromocytoma cell line, during depolarization or exposure to bradykinin or cAMP. However, to our knowledge transactivation of EGFRs has not previously been reported in primary cultures of neurons. The signal pathway inducing "shedding" of growth factors is complex and may be cell type-, G-protein coupled receptor-, and developmental stage-specific.
A significant portion of K~+-mediated ERK_(1/2) phosphorylation depended upon NMDA receptor activation, as indicated by its inhibition by NMDA, an antagonist of the NMDA receptor. ERK_(1/2) phosphorylation in cerebellar granule cells in response to NMDA receptor stimulation has previously been demonstrated by Sato et al. and Zhu et al., but these authors did not investigate whether transactivation was involved.
In the present cultures the initial K~+-mediated glutamate release amounts to about 10 nmol/min per mg protein, but due to a time-dependent reduction of release and to concentration-dependent reuptake the glutamate concentration in the medium is only 10-15 nmol/mg protein after 30 min of incubation. With approximately 1 mg cell protein incubated in 2 ml of medium, this amount corresponds to a medium glutamate concentration of 5-8 uM, and perhaps more in the synaptic clefts. This concentration is likely to suffice for activation of NMDA receptors, which have a high affinity for glutamate, but perhaps not for AMPA receptors which have a much lower affinity. This may explain why CNQX, an antagonist at AMPA receptors, had no effect, whereas Wu et al. and Limatola et al., using addition of receptor agonists have demonstrated ERK_(1/2) phosphorylation induced by AMPA receptor activation in cerebellar granule cells. Again, it was not investigated whether transactivation was involved.
The present report together with our previous paper show a remarkable complexity of the signaling pathways between K~+-mediated depolarization of cerebellar granule cells in primary cultures and ERK_(1/2) phosphorylation . They include a glutamate-induced ERK_(1/2) phosphorylation secondary to K~+-evoked glutamate release at both 20 and 60 min, which is dependent upon EGFR transactivation. The present study has shown that only the former of these two increases in ERK_(1/2) phosphorylation requires PKC activity. The PKC dependence of the response to glutamate after 20 min was confirmed by addition of 50 uM glutamate to glutamine-free medium, although a minor part of this effect appeared to be PKC-independent. In addition the K+-mediated depolarization per se causes ERK_(1/2) phosphorylation at the same time points. However, this phosphorylation occurs independently of transactivation and is also independent of PKC activation, at least at 20 min. PKC inhibition was not tested in glutamine-free medium at 60 min but there was no indication of any PKC-dependent component at 60 min in the glutamine-replete medium.
Accumulating evidence suggests that ERK phosphorylation may play an important role in at least certain types of long-term potentiation (LTP) and learning. Recently, NMDA- and PKC-dependent LTP has been demonstrated at the cerebellar mossy fiber-granule cell synapse. Also, investigation of cytosolic [Ca~(2+)] dynamics during repetitive neurotransmission in cerebellar slices showed that glutamate receptor-mediated [Ca~(2+)]j increase in granule cell dendrites was locally amplified by opening of voltage-dependent Ca~(2+) channels and Ca~(2+) release from intracellular stores. Moreover, granule cell firing contributed to elevating cytosolic [Ca~(2+)] through voltage-dependent channels also in the dendrites that were not synaptically stimulated. These findings attest to the importance of both glutamate-mediated and depolarization-mediated ERK phosphorylation in cerebellar granule cells but it is not known where PKC activity fits into the picture.
Conclusion
In conclusion, depolarization of 7-8-day old cerebellar granule neurons, obtained from 7-day-old mice, by exposure to 45 mM extracellular [K~+]_e leads to phosphorylation of ERK_(1/2), mediated by NMDA receptor activation and partly by a transactivation process, leading to release of HB-EGF and perhaps also of TGF-α. The subsequent action of these EGFR agonists may play a major role for the effects of NMDA receptor activation on brain plasticity.
引文
1 Seroogy KB, Gall CM, Lee DC, et al. Proliferative zones of postnatal rat brain express epidermal growth factor receptor mRNA. Brain Res. 1995; 670: 157-164.
2 Carrasco E, Blum M, Weickert CS, et al. Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone. Brain Res Dev Brain Res. 2003; 140: 1-13.
3 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 1997; 272:19103-19106.
4 Thiels E, Kanterewicz BI, Norman ED, et al. Long-term depression in the adult hippocampus in vivo involves activation of extracellular signal-regulated kinase and phosphorylation of Elk-1. J Neurosci. 2002; 22: 2054-2062.
5 Runyan JD, Moore AN, Dash PK. A role for prefrontal cortex in memory storage for trace fear conditioning. J Neurosci. 2004; 24: 1288-1295.
6 Baron C, Benes C, Van Tan H, et al. Potassium chloride pulse enhances mitogen-activated protein kinase activity in rat hippocampal slices. J Neurochem. 1996; 66: 1005-1010.
7 Cavanaugh JE, Ham J, Herman M, et al. Differential regulation of mitogen-activated protein kinases ERK1/2 and ERK5 by neurotrophins, neuronal activity, and cAMP in neurons. J Neurosci. 2001; 21: 434-443.
8 Nashat AH, Langer R. Temporal characteristics of activation, deactivation, and restimulation of signal transduction following depolarization in the pheochromocytoma cell line PC12. Mol Cell Biol. 2003; 23: 4788-4795.
9 Corvol JC, Valjent E, Toutant M, et al. Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices. J Biol Chem.2005; 280: 660-668.
10 Schwarzschild MA, Cole RL, Meyers MA, et al. Contrasting calcium dependencies of SAPK and ERK activations by glutamate in cultured striatal neurons. J Neurochem. 1999; 72: 2248-2255.
11 Vanhoutte P, Barnier JV, Guibert B, et al. Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices. Mol Cell Biol.1999; 19: 136-146.
12 Chandler LJ, Sutton G, Dorairaj NR, et al. N-methyl-D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures. J Biol Chem. 2001; 276: 2627-2636
13 Fuller G, Veitch K, Ho LK, et al. Activation of p44/p42 MAP kinase in striatal neurons via kainate receptors and PI3 kinase. Brain Res Mol Brain Res. 2001; 89:126-132.
14 Wu X, Zhu D, Jiang X, et al. AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression. J Neurochem. 2004; 90: 807-818.
15 Rakhit S, Clark CJ, O'shaughnessy CT, et al. N-methyl-D-aspartate and brain-derived neurotrophic factor induce distinct profiles of extracellular signal-regulated kinase, mitogen-and stress-activated kinase, and ribosomal s6 kinase phosphorylation in cortical neurons. Mol Pharmacol. 2005; 67:1158-1165.
16 Balazs R, Gallo V, et al. Effect of depolarization on the maturation of cerebellar granule cells in culture. Brain Res. 1988; 468: 269-276.
17 Palaiologos G, Hertz L, Schousboe A. Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate. J Neurochem. 1988; 51: 317-320.
18 Palaiologos G, Hertz L, Schousboe A. Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem Res. 1989; 14: 359-366.
19 Peng LA, Juurlmk BH, Hertz L. Differences in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence and in the absence of a depolarizing potassium concentration. Brain Res Dev Brain Res. 1991; 63: 1-12.
20 Peng LA, Schousboe A, Hertz L. Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells. Neurochem Res. 1991; 16: 29-34.
21 Juurlink BH, Hertz L. Ischemia-induced death of astrocytes and neurons in primary culture: pitfalls in quantifying neuronal cell death. Brain Res Dev Brain Res. 1993; 71: 239-246.
22 Hogstad S, Svenneby G, Torgner IA, et al. Glutaminase in neurons and astrocytes cultured from mouse brain: kinetic properties and effects of phosphate, glutamate, and ammonia.Neurochem Res. 1988; 13: 383-388.
23 Cox JA, Felder CC, Henneberry RC. Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons. Neuron. 1990; 4: 941-947.
24 Zhao Z, Peng L. Glutamate effects on calcium homeostasis in cerebellar granule cells in primary cultures grown under depolarizing and nondepolarizing conditions.Synapse. 1993; 13: 315-321.
25 Benveniste H, Jorgensen MB, Sandberg M, et al. Ischemic damage in hippocampal CA1 is dependent on glutamate release and intact innervation from CA3. J Cereb Blood Flow Metab. 1989; 9: 629-639.
26 Erecinska M, Silver IA. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol. 1990; 35: 245-296.
27 Norenberg MD, Martinez-Hernandez A. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 1979; 161: 303-310.
28 Hogstad S, Svenneby G, Torgner IA, et al. Glutaminase in neurons and astrocytes cultured from mouse brain: kinetic properties and effects of phosphate, glutamate, and ammonia. Neurochem Res. 1988; 13: 383-388.
29 Hertz L, Yu AC, Schousboe A. Uptake and metabolism of malate in neurons and astrocytes in primary cultures. J Neurosci Res. 1992; 33: 289-296.
30 Victoria C, Thomas K. Metabotropic Glutamate Receptors: Electrical and Chemical Signaling Properties. Neuroscientist. 2002; 8: 551-561
31 Bishayee S. Role of conformational alteration in the epidermal growth factor receptor (EGFR) function.Biochem Pharmacol. 2000; 60: 1217-1223.
32 Harris DA, Walter NG. Probing RNA structure and metal-binding sites using terbium (III) footprinting.Curr Protoc Nucleic Acid Chem. 2003; Chapter 6: Unit 6.8.
33 Birecree E, King LE Jr, Nanney LB. Epidermal growth factor and its receptor in the developing human nervous system. Brain Res Dev Brain Res. 1991; 60: 145-154
34 Nakagawa T, Sasahara M, Hayase Y, et al. Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat. Dev. Brain Res. 1998; 108: 263-272.
35 Falk A, Frisen J. Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res. 2002; 69: 757-62.
36 Carrasco E, Blum M, Weickert CS, et al. Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone. Brain Res Dev Brain Res. 2003; 140: 1-13.
37 Lu YC, Qi JG, Wang TH, et al. Distribution of epidermal growth factor in central nervous system of adult rhesus monkey. Sichuan Da Xue Xue Bao Yi Xue Ban. 2005; 36: 618-621.
38 Gu L, Li B, Yang X, et al. Depolarization-induced, glutamate Receptor-mediated and Transactivation-dependent Extracellular-signal Regulated Kinase Phosphorylation in Cultured Cerebellar Granule Neurons. Neurosci. 2007; 147: 342-353
39 Opanashuk LA, Mark RJ, Porter J, et al. Heparin-binding epidermal growth factor-like growth factor in hippocampus: modulation of expression by seizures and anti-excitotoxic action. J Neurosci. 1999; 19:133-146.
40 Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr. Relat. Cancer. 2001; 8: 11-31.
41 Krainock R, Murphy S. Heregulin upregulates the expression of nitric oxide synthase NOS-1 in rat cerebellar granule neurons via the ErbB4 receptor. J Neurochem. 2001; 76: 312-315.
42 Lezama R, Ortega A, Ordaz B, et al. Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons. J Neurochem. 2005; 93: 1189-1198.
43 Law AJ, Shannon WC, Hyde TM, et al. Neuregulin-1 NRG-1 mRNA and protein in the adult human brain. Neurosci. 2004; 127: 125-136.
44 Beerli RR, Hynes NE. Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J Biol Chem. 1996; 271: 6071-6076.
45 Huang YZ, Wang Q, Xiong WC, et al. Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95. J Biol Chem. 2001; 276: 19318-19326.
46 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 1997; 272: 19103-19106.
47 Yamaguchi H, Wang HG. The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene. 2001; 20: 7779-7786.
48 Xue L, Murray JH, Tolkovsky AM. The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J Biol Chem. 2000; 275: 8817-8824.
49 Bruno V, Battaglia G., Casabona G, et al. Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta. J Neurosci. 1998; 18: 9594-9600.
50 Daub H., Wallasch C, Lankenau A, et al. Signal characteristics of G protein transactivated EGF receptor. EMBO J. 1997; 16: 7032-7044.
51 Rhee SG, Choi KD, Regulation of inositol phospholipidspecific phospholipase C isozymes. J Biol Chem. 1992; 267:12393-12396.
52 Dennis EA, Rhee SG, Billah MM, et al. Role of phospholipases in generating lipid second messengers in signal transduction. FASEBJ. 1991; 5: 2068-2077.
53 53 Cockcroft S, Thomas GMH, Inositol-lipid-specific phospholipase C isoenzymes and their differential regulation by receptors. Biochem J. 1992; 288: 1-14.
54 Benavides M, Laorden ML, Marin MT, et al. Role of PKC-alpha, gamma isoforms in regulation of c-Fos and TH expression after naloxone-induced morphine withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups. J Neurochem. 2005; 95: 1249-1258.
55 Naoaki S, Yasuhito S . Protein Kinase C (PKC ) : Function of Neuron Specific Isotyp. J Biochem. 2002; 132: 683-687.
56 Lindroth P, Mopper K. High performance liquid chromatography determination of subpicomole amounts of a mino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Analytical Chemistry. 1979; 51: 1667-16 74.
57 Yu AC, Schousboe A, Hertz L. Metabolic fate of (14C) -lablled glutamate in astrocytes. J Neurochem. 1982; 39: 954-966.
58 Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.
59 Marjou A, Delouvee A, Thiery JP, et al. Involvement of epidermal growth factor receptor in chemically induced mouse bladder tumour progression. Carcinogenesis . 2000; 21: 2211-2218.
60 Li B, Du T, Li H, et al. Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons.Br J Pharmacol. 2008; 154:191-203.
61 Falk A F. J Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res. 2002; 69: 757-762.
62 Traverse S., Seedorf K., Paterson H, et al. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol. 1994; 4: 694-701.
63 Goishi K, Higashiyama S, Klagsbrun M, et al. Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity. Mol Biol Cell. 1995; 6: 967-980.
64 Umata T, Hirata M, Takahashi T, et al. A dual signaling cascade that regulates the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. J Biol Chem. 2001; 276: 30475-30482.
65 Dethlefsen SM, Raab G, Moses, et al. Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. J Cell Biochem. 1998; 69: 143-153.
66 Kalmes A, Vesti BR., Daum G, et al. Heparin blockage of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor EGF receptor transactivation by heparin-binding EGF-like growth factors. Circ Res. 2000; 87: 92-98.
67 Zwick E, Daub H, Aoki N, et al. Critical role of calcium- dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem. 1997; 272: 24767-24770.
68 Grewal JS, Luttrell LM., Raymond JR. G protein-coupled receptors desensitize and down-regulate epidermal growth factor recptor in renal mesangial cells. J Biol Chem. 2001; 276: 27335-27344.
69 Tebar F, Llado A, Enrich C. Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC. FEBS Lett. 2002; 517: 206-210.
70 Cussac D, Schaak S, Gales C, et al. alpha (2B) -Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells. Am J Physiol Renal Physiol. 2002; 282: F943-952.
71 Luo J. The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex. Cerebellum. 2005; 4: 239-245.
72 Gursoy-Ozdemir Y, Qiu J, Matsuoka N, et al. Cortical spreading depression activates and upregulates MMP-9. J Clin Invest. 2004; 113: 1447-1455.
73 Shah BH, Farshori MP, Jambusaria A, et al. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem. 2003; 278: 19118-19126.
74 Shah BH, Farshori MP, Catt KJ. Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor. J Biol Chem. 2004; 279: 414 -420.
75 Shah BH, Neithardt A, Chu DB, et al. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J Cell Physiol. 2006; 206: 47-57.
76 Piiper A, Dikic I, Lutz MP, et al. Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells. J Biol Chem. 2002; 277: 43623-43630.
77 Baron C, Benes C, Van Tan H, et al. Potassium chloride pulse enhances mitogen-activated protein kinase activity in rat hippocampal slices. J Neurochem. 1996. 66:1005-1010.
78 Corvol JC, Valjent E, Toutant M, et al. Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices. J Biol Chem. 2005; 280: 660-668.
79 Vanning T, Christopherson P, Schousboe A, et al. Pharmacological characterisation of voltage-sensitive calcium channels and neurotransmitter release from mouse cerebellar granule cells in culture. J Neurosci Res. 1997; 48: 43-52.
80 English JD, Sweatt JD. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem. 1996; 271: 24329-24332.
81 Yuan LL, Adams JP, Swank M, et al. Protein kinase modulation of dendriticK+ channels in hippocampus involves a mitogenactivated protein kinase pathway. J Neurosci. 1996; 22: 4860-4868.
82 Balazs R. Trophic effect of glutamate. Curr Top Med Chem. 2006; 6: 961-968.
83 Atkins CM, Selcher JC, Petraitis JJ, et al. The MAPK cascade is required for mammalian associative learning. Nat Neurosci. 1998; 1: 602-609.
84 Winder DG, Martin KC, Muzzio IA, et al. ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors. Neuron. 1999; 24: 715-726.
85 Dudek SM, Fields RD. Mitogen-activated protein kinase/ extracellular signal-regulated kinase activation in somatodendritic compartments: roles of action potentials, frequency, and mode of calcium entry. J Neurosci. 2001; 21: 1-5.
86 Huber KM, Mauk MD, Thompson C, et al. A critical period of protein kinase activity after tetanic stimulation is required for the induction of long-term potentiation. Learn Mem. 1995; 2: 81-100.
87 D'Angelo E, Rossi P, Gall D, et al. Long-term potentiation of synaptic transmission at the mossy fiber-granule cell relay of cerebellum. Prog Brain Res. 2005; 148: 69-80.
88 Nieus T, Sola E, Mapelli J, et al. LTP regulates burst initiation and frequency at mossy fiber-granule cell synapses of rat cerebellum: experimental observations and theoretical predictions. J Neurophysiol. 2006; 95: 686-699.
89 Mapelli J, D'Angelo E. The spatial organization of longterm synaptic plasticity at the input stage of cerebellum. J Neurosci. 2007; 27: 1285-1296.
90 Gall D, Prestori F, Sola E, et al. Intracellular calcium regulation by burst discharge determines bi-directional long-term synaptic plasticity at the cerebellum input stage. J Neurosci. 2007; 25: 4813-4822.
1 Pandiella A, Massague J. Cleavage of the membrane precursor for transforming growth factor alpha is a regulated process. Proc Natl Acad Sci U S A. 1991; 88:1726-1730
2 Harris DA, Walter NG. Probing RNA structure and metal-binding sites using terbium (III) footprinting. Curr Protoc Nucleic Acid Chem. 2003; Chapter 6: Unit 6.8.
3 Birecree E, King LE Jr, Nanney LB . Epidermal growth factor and its receptor in the developing human nervous system. Brain Res Dev Brain Res. 1991; 60: 145-154
4 Nakagawa T, Sasahara M, Hayase Y, et al. Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat. Dev. Brain Res. 1998; 108: 263-272.
5 Falk A, Frisen J . Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res. 2002; 69: 757-762.
6 Carrasco E, Blum M, Weickert CS, et al. Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone. Brain Res Dev Brain Res. 2003; 140: 1-13.
7 Lu YC, Qi JG, Wang TH, et al. Distribution of epidermal growth factor in central nervous system of adult rhesus monkey. Sichuan Da Xue Xue Bao Yi Xue Ban. 2005; 36: 618-621.
8 Higashiyama S, Abraham JA, Miller J, et al. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991; 251: 936-939.
9 Jin K, Mao X, Sun Y, Xie L, et al. Heparine-binding epidermal growth factor-like growth factor: hypoxia-inducible expression in vitro and stimulation of neurogenesis in vitro and in vivo. J Neurosci. 2002; 22: 5365-5373.
10 Kornblum H, Zurcher S, Werb Z, et al. Multiple trophic actions of heparin-binding epidermal growth factor HB-EGF in the central nervous system. Eur. J. Neurosci. 1999; 11: 3236-3246.
11 Opanashuk L, Mark R, Porter J, et al. Heparin-binding epidermal growth factor-like growth factor in hippocampus: modulation of expression by seizures and anti-excitotoxic action. J Neurosci 1999; 19: 133-146.
12 Nakagawa T, Sasahara M, Hayase Y, et al. Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat. Dev Brain Res 1998; 108:263-272.
13 Grewal, J, Luttrell L, Raymond J . G protein-coupled receptors desensitize and down-regulate epidermal growth factor recptor in renal mesangial cells. J Biol Chem. 2001; 276: 27335-27344.
14 Tanaka N, Sasahara M, Ohno M, et al. Heparin-binding epidermal growth factor-like growth factor mRNA expression in neonatal rat brain with hypoxic/ischemic injury. Brain Res. 1999; 827: 130-138.
15 Gu L, Li B, Yang X, Hu X, et al. Depolarization-induced, glutamate Receptor-mediated and Transactivation-dependent Extracellular-signal Regulated Kinase Phosphorylation in Cultured Cerebellar Granule Neurons. Neurosci. 2007; 147: 342-353
16 Li B, Du T, Li H, et al. Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons.Br J Pharmacol. 2008; 154: 191-203.
17 Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer. 2001; 8: 11-31.
18 Krainock R, Murphy S . Heregulin upregulates the expression of nitric oxide synthase NOS-1 in rat cerebellar granule neurons via the ErbB4 receptor. J Neurochem. 2001; 76: 312-315.
19 Lezama R, Ortega A, Ordaz B, et al. Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons. J Neurochem. 2005; 93: 1189-1198.
20 Pinkas-Kramarski, R, Eilam, R, Alroy, I, et al. Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation. Oncogene. 1997; 15:2803-2815.
21 Huang, Y, Wang, Q, Xiong W.C, et al. Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95. J Biol Chem. 2001; 276: 19318-19326.
22 Beerli R, Hynes NE . Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J Biol Chem. 1996; 15 : 6071-6076.
23 Jones F, Golding J, Gassmann M. ErbB4 signaling during breast and neural development: novel genetic models reveal unique ErbB4 activities.Cell Cycle. 2003; 2: 555-559.
24 Law A, Shannon Weickert C, Hyde TM, et al. Neuregulin-1 NRG-1 mRNA and protein in the adult human brain. Neurosci. 2004; 127:125-136.
25 Rio C, Rieff HI, Qi P, et al. Neuregulin and ErbB receptors play a critical role in neuronal migration.Neuron. 1997; 19: 39-50.
26 Daub H, Wallasch C, Lankenau A, et al. Signal characteristics of G protein transactivated EGF receptor. EMBO J. 1997; 16: 7032-7044.
27 Peavy R, Chang M, Sanders B, et al. Metabotropic glutamate receptor 5-induced phosphorylation of extracellular signal-regulated kinase in astrocytes depends on transactivation of the epidermal growth factor receptor. J Neurosci. 2001; 21: 9619-9628.
28 Kalmes A, Vesti B, Daum G, et al. Heparin blockage of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor EGF receptor transactivation by heparin-binding EGF-like growth factors. Circ Res. 2000; 87: 92-98.
29 Saito S, Frank G, Motley E, et al. Metalloprotease inhibitor blocks angiotensin -induced migration through inhibition of epidermal growth factor receptor transactivation. Biochem Biophys Res Commun. 2002; 294: 1023-1029.
30 Goishi K, Higashiyama S, Klagsbrun M, et al. Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity. Mol Biol Cell. 1995; 6: 967-980.
31 Umata T, Hirata M, Takahashi T, et al. A dual signaling cascade that regulates the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. J. Biol. Chem. 2001; 276: 30475-30482
32 Izumi Y, Hirata M, Hasuwa H,A metalloprotease-disintegrin, MDC9/meltrin-gamma/ ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J . 1998; 17: 7260-7272.
33 Dethlefsen S, Raab, G, Moses, et al. Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. J Cell Biochem. 1998; 69:143-153.
34 Zwick E, Daub H, Aoki N, et al. Critical role of calcium- dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem 1997; 272: 24767-24770.
35 Tebar F, Llado A, Enrich C, et al. Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC. FEBS Lett. 2002; 517: 206-210.
36 Cussac D, Schaak S, Denis C, et al. Alpha 2B-adrenergic receptor activates MAPK via a pathway involving arachidonic acid metabolism, matrix metalloproteinases, and epidermal growth factor receptor transactivation. J Biol Chem. 2002; 277:19882-198828.
37 Cussac D, Schaak S, Gales C, et al. Flordellis C, Denis C, Paris H.alpha (2B) -Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells. Am J Physiol Renal Physiol. 2002; 282: 943-952.
38 Pierce K, Tohgo A, Ahn S, et al. Epidermal growth factor EGF receptor-dependent ERK activation by G protein-coupled receptors. J Biol Chem. 2001; 276: 23155-23160.
39 Bruno V, Battaglia G, Casabona G, et al. Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta. J. Neurosci. 1998; 18: 9594-9600.
40 Zelenaia O, Schlag B, Gochenauer G, et al. Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. Mol. Pharmacol. 2000; 57: 667-678.
41 Xue L, Murray J, Tolkovsky A. The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J Biol Chem. 2000; 275: 8817-8824.
42 Shah BH, Farshori M, Jambusaria A, et al. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation.J Biol Chem. 2003; 278: 19118-191126.
43 Shah BH, Farshori MP, Catt KJ. Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor. J Biol Chem. 2004; 279: 414-420.
44 Shah BH, Neithardt A, Chu DB, et al. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J Cell Physiol. 2006; 206: 47-57.
45 Piiper A, Dikic I, Lutz MP, et al. Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells. J Biol Chem. 2002; 277: 43623-43630.
46 Shah BH, Farshori MP, Jambusaria A, et al. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERKl/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem. 2003; 278: 19118-19126.
47 Boonstra J, De Laat SW, Ponec M. Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes. Exp Cell Res. 1985; 161: 421-433.
48 Traverse, S., Seedorf, K., Paterson, H., et al. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol. 1994; 4; 694-701.
49 49 Soltoff SP, Cantley LC. 120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC 12 and other cells. J Biol Chem. 1996; 271: 563-567.
50 Klesse LJ, Meyers KA, Marshall CJ, et al. Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells. Oncogene. 1999; 18: 2055-2068.
51 Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995; 267: 2003-2006.
52 Jackson, T.R., Blader, I.J., Hammonds-Odie, L.P., et al. Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase. J Cell. Sci. 1996; 109: 289-300.
53 Kimura, K., Hattori, S., Kabuyama, Y., et al. Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 1994; 269: 18961-18967.
54 Posern, G., Saffrich, R., Ansorge, W., et al. Rapid lamellipodia formation in nerve growth factor-stimulated PC12 cells is dependent on Rac and PI3K activity. J Cell Physiol. 2000; 183; 416-424.
55 Yasui H, Katoh H., Yamaguchi Y, et al. Differential responses to nerve growth factor and epidermal growth factor in neurite outgrowth of PC12 cells are determined by Racl activation systems. J Biol Chem. 2001; 276: 15298-15305.
56 Corbit KC, Soh JW, Yoshida K, et al. Different protein kinase C isoforms determine growth factor specificity in neuronal cells.Mol Cell Biol. 2000; 20: 5392-5403.
57 Lev, S, Moreno H, Martinez R, et al. Protein tyrosine kinase PYK2 involved in Ca (2+) -induced regulation of ion channel and MAP kinase functions. Nature. 1995; 376: 737-745.
58 Coogan AN, Piggins HD. Circadian and photic regulation of phosphorylation of ERK1/2 and Elk-1 in the suprachiasmatic nuclei of the Syrian hamster. J Neurosci. 2003; 23: 3085-3093.
59 Kuroda KO, Meaney MJ, Uetani N, et al. ERK-FosB signaling in dorsal MPOA neurons plays a major role in the initiation of parental behavior in mice. Mol Cell Neurosci. 2007; 36: 121-131.
60 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 1997; 272: 19103-19106.
61 Di Segni A, Farin K, Pinkas-Kramarski R. ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells: involvement of PI3K and Erk signaling. J Mol Neurosci. 2006; 29: 257-267.
62 Jin K, Sun Y, Xie L, et al. Post-ischemic administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) reduces infarct size and modifies neurogenesis after focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 2004; 24: 399-408.
63 Michalsky M, Kuhn A, Mehta V, et al. Heparin-binding EGF-like growth factor decreases apotosis in intestinal epithelial cells in vitro. J Pediatr Surg. 2001; 36: 1130-1135.
64 Lara-Marquez M, Mehta V, Michalsky M, et al. Heparin-binding EGF-like growth factor down regulates proinflammatory cytokin-induced nitric oxide and inducible nitric oxide synthase production in intestinal epithelial cells. Nitric Oxide. 2005; 6: 142-152.
65 Bryson, J, Coy P, Gottlob K, et al. Increased hexokinase activity, or either ectopic or endogenous origin, protects renal epithelial cells against acute oxidant-induced cell death. J Biol Chem. 2002; 277: 11392-11400.
66 Zhu Y, Yang G, Ahlemeyer B, et al. Transforming growth factor-beta 1 increases bad phosphorylation and protects neurons against damage. J Neurosci. 2002; 22: 3898-3909.
67 Han B, Holtzman D . BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci. 2000; 20: 5775-5781.
68 Lenhard T, Schober A, Suter-Crazzolara C, et al. Fibroblast growth factor-2 requires glial-cell-line-derived neurotrophic factor for exerting its neuroprotective actions on glutamate-lesioned hippocampal neurons. Mol Cell Neurosci. 2002; 20: 181-197.
2 Carrasco E, Blum M, Weickert CS, et al. Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone. Brain Res Dev Brain Res. 2003; 140: 1-13.
3 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 1997; 272:19103-19106.
4 Thiels E, Kanterewicz BI, Norman ED, et al. Long-term depression in the adult hippocampus in vivo involves activation of extracellular signal-regulated kinase and phosphorylation of Elk-1. J Neurosci. 2002; 22: 2054-2062.
5 Runyan JD, Moore AN, Dash PK. A role for prefrontal cortex in memory storage for trace fear conditioning. J Neurosci. 2004; 24: 1288-1295.
6 Baron C, Benes C, Van Tan H, et al. Potassium chloride pulse enhances mitogen-activated protein kinase activity in rat hippocampal slices. J Neurochem. 1996; 66: 1005-1010.
7 Cavanaugh JE, Ham J, Herman M, et al. Differential regulation of mitogen-activated protein kinases ERK1/2 and ERK5 by neurotrophins, neuronal activity, and cAMP in neurons. J Neurosci. 2001; 21: 434-443.
8 Nashat AH, Langer R. Temporal characteristics of activation, deactivation, and restimulation of signal transduction following depolarization in the pheochromocytoma cell line PC12. Mol Cell Biol. 2003; 23: 4788-4795.
9 Corvol JC, Valjent E, Toutant M, et al. Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices. J Biol Chem.2005; 280: 660-668.
10 Schwarzschild MA, Cole RL, Meyers MA, et al. Contrasting calcium dependencies of SAPK and ERK activations by glutamate in cultured striatal neurons. J Neurochem. 1999; 72: 2248-2255.
11 Vanhoutte P, Barnier JV, Guibert B, et al. Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices. Mol Cell Biol.1999; 19: 136-146.
12 Chandler LJ, Sutton G, Dorairaj NR, et al. N-methyl-D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures. J Biol Chem. 2001; 276: 2627-2636
13 Fuller G, Veitch K, Ho LK, et al. Activation of p44/p42 MAP kinase in striatal neurons via kainate receptors and PI3 kinase. Brain Res Mol Brain Res. 2001; 89:126-132.
14 Wu X, Zhu D, Jiang X, et al. AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression. J Neurochem. 2004; 90: 807-818.
15 Rakhit S, Clark CJ, O'shaughnessy CT, et al. N-methyl-D-aspartate and brain-derived neurotrophic factor induce distinct profiles of extracellular signal-regulated kinase, mitogen-and stress-activated kinase, and ribosomal s6 kinase phosphorylation in cortical neurons. Mol Pharmacol. 2005; 67:1158-1165.
16 Balazs R, Gallo V, et al. Effect of depolarization on the maturation of cerebellar granule cells in culture. Brain Res. 1988; 468: 269-276.
17 Palaiologos G, Hertz L, Schousboe A. Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate. J Neurochem. 1988; 51: 317-320.
18 Palaiologos G, Hertz L, Schousboe A. Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem Res. 1989; 14: 359-366.
19 Peng LA, Juurlmk BH, Hertz L. Differences in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence and in the absence of a depolarizing potassium concentration. Brain Res Dev Brain Res. 1991; 63: 1-12.
20 Peng LA, Schousboe A, Hertz L. Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells. Neurochem Res. 1991; 16: 29-34.
21 Juurlink BH, Hertz L. Ischemia-induced death of astrocytes and neurons in primary culture: pitfalls in quantifying neuronal cell death. Brain Res Dev Brain Res. 1993; 71: 239-246.
22 Hogstad S, Svenneby G, Torgner IA, et al. Glutaminase in neurons and astrocytes cultured from mouse brain: kinetic properties and effects of phosphate, glutamate, and ammonia.Neurochem Res. 1988; 13: 383-388.
23 Cox JA, Felder CC, Henneberry RC. Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons. Neuron. 1990; 4: 941-947.
24 Zhao Z, Peng L. Glutamate effects on calcium homeostasis in cerebellar granule cells in primary cultures grown under depolarizing and nondepolarizing conditions.Synapse. 1993; 13: 315-321.
25 Benveniste H, Jorgensen MB, Sandberg M, et al. Ischemic damage in hippocampal CA1 is dependent on glutamate release and intact innervation from CA3. J Cereb Blood Flow Metab. 1989; 9: 629-639.
26 Erecinska M, Silver IA. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol. 1990; 35: 245-296.
27 Norenberg MD, Martinez-Hernandez A. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 1979; 161: 303-310.
28 Hogstad S, Svenneby G, Torgner IA, et al. Glutaminase in neurons and astrocytes cultured from mouse brain: kinetic properties and effects of phosphate, glutamate, and ammonia. Neurochem Res. 1988; 13: 383-388.
29 Hertz L, Yu AC, Schousboe A. Uptake and metabolism of malate in neurons and astrocytes in primary cultures. J Neurosci Res. 1992; 33: 289-296.
30 Victoria C, Thomas K. Metabotropic Glutamate Receptors: Electrical and Chemical Signaling Properties. Neuroscientist. 2002; 8: 551-561
31 Bishayee S. Role of conformational alteration in the epidermal growth factor receptor (EGFR) function.Biochem Pharmacol. 2000; 60: 1217-1223.
32 Harris DA, Walter NG. Probing RNA structure and metal-binding sites using terbium (III) footprinting.Curr Protoc Nucleic Acid Chem. 2003; Chapter 6: Unit 6.8.
33 Birecree E, King LE Jr, Nanney LB. Epidermal growth factor and its receptor in the developing human nervous system. Brain Res Dev Brain Res. 1991; 60: 145-154
34 Nakagawa T, Sasahara M, Hayase Y, et al. Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat. Dev. Brain Res. 1998; 108: 263-272.
35 Falk A, Frisen J. Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res. 2002; 69: 757-62.
36 Carrasco E, Blum M, Weickert CS, et al. Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone. Brain Res Dev Brain Res. 2003; 140: 1-13.
37 Lu YC, Qi JG, Wang TH, et al. Distribution of epidermal growth factor in central nervous system of adult rhesus monkey. Sichuan Da Xue Xue Bao Yi Xue Ban. 2005; 36: 618-621.
38 Gu L, Li B, Yang X, et al. Depolarization-induced, glutamate Receptor-mediated and Transactivation-dependent Extracellular-signal Regulated Kinase Phosphorylation in Cultured Cerebellar Granule Neurons. Neurosci. 2007; 147: 342-353
39 Opanashuk LA, Mark RJ, Porter J, et al. Heparin-binding epidermal growth factor-like growth factor in hippocampus: modulation of expression by seizures and anti-excitotoxic action. J Neurosci. 1999; 19:133-146.
40 Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr. Relat. Cancer. 2001; 8: 11-31.
41 Krainock R, Murphy S. Heregulin upregulates the expression of nitric oxide synthase NOS-1 in rat cerebellar granule neurons via the ErbB4 receptor. J Neurochem. 2001; 76: 312-315.
42 Lezama R, Ortega A, Ordaz B, et al. Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons. J Neurochem. 2005; 93: 1189-1198.
43 Law AJ, Shannon WC, Hyde TM, et al. Neuregulin-1 NRG-1 mRNA and protein in the adult human brain. Neurosci. 2004; 127: 125-136.
44 Beerli RR, Hynes NE. Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J Biol Chem. 1996; 271: 6071-6076.
45 Huang YZ, Wang Q, Xiong WC, et al. Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95. J Biol Chem. 2001; 276: 19318-19326.
46 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 1997; 272: 19103-19106.
47 Yamaguchi H, Wang HG. The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene. 2001; 20: 7779-7786.
48 Xue L, Murray JH, Tolkovsky AM. The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J Biol Chem. 2000; 275: 8817-8824.
49 Bruno V, Battaglia G., Casabona G, et al. Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta. J Neurosci. 1998; 18: 9594-9600.
50 Daub H., Wallasch C, Lankenau A, et al. Signal characteristics of G protein transactivated EGF receptor. EMBO J. 1997; 16: 7032-7044.
51 Rhee SG, Choi KD, Regulation of inositol phospholipidspecific phospholipase C isozymes. J Biol Chem. 1992; 267:12393-12396.
52 Dennis EA, Rhee SG, Billah MM, et al. Role of phospholipases in generating lipid second messengers in signal transduction. FASEBJ. 1991; 5: 2068-2077.
53 53 Cockcroft S, Thomas GMH, Inositol-lipid-specific phospholipase C isoenzymes and their differential regulation by receptors. Biochem J. 1992; 288: 1-14.
54 Benavides M, Laorden ML, Marin MT, et al. Role of PKC-alpha, gamma isoforms in regulation of c-Fos and TH expression after naloxone-induced morphine withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups. J Neurochem. 2005; 95: 1249-1258.
55 Naoaki S, Yasuhito S . Protein Kinase C (PKC ) : Function of Neuron Specific Isotyp. J Biochem. 2002; 132: 683-687.
56 Lindroth P, Mopper K. High performance liquid chromatography determination of subpicomole amounts of a mino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Analytical Chemistry. 1979; 51: 1667-16 74.
57 Yu AC, Schousboe A, Hertz L. Metabolic fate of (14C) -lablled glutamate in astrocytes. J Neurochem. 1982; 39: 954-966.
58 Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.
59 Marjou A, Delouvee A, Thiery JP, et al. Involvement of epidermal growth factor receptor in chemically induced mouse bladder tumour progression. Carcinogenesis . 2000; 21: 2211-2218.
60 Li B, Du T, Li H, et al. Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons.Br J Pharmacol. 2008; 154:191-203.
61 Falk A F. J Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res. 2002; 69: 757-762.
62 Traverse S., Seedorf K., Paterson H, et al. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol. 1994; 4: 694-701.
63 Goishi K, Higashiyama S, Klagsbrun M, et al. Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity. Mol Biol Cell. 1995; 6: 967-980.
64 Umata T, Hirata M, Takahashi T, et al. A dual signaling cascade that regulates the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. J Biol Chem. 2001; 276: 30475-30482.
65 Dethlefsen SM, Raab G, Moses, et al. Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. J Cell Biochem. 1998; 69: 143-153.
66 Kalmes A, Vesti BR., Daum G, et al. Heparin blockage of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor EGF receptor transactivation by heparin-binding EGF-like growth factors. Circ Res. 2000; 87: 92-98.
67 Zwick E, Daub H, Aoki N, et al. Critical role of calcium- dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem. 1997; 272: 24767-24770.
68 Grewal JS, Luttrell LM., Raymond JR. G protein-coupled receptors desensitize and down-regulate epidermal growth factor recptor in renal mesangial cells. J Biol Chem. 2001; 276: 27335-27344.
69 Tebar F, Llado A, Enrich C. Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC. FEBS Lett. 2002; 517: 206-210.
70 Cussac D, Schaak S, Gales C, et al. alpha (2B) -Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells. Am J Physiol Renal Physiol. 2002; 282: F943-952.
71 Luo J. The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex. Cerebellum. 2005; 4: 239-245.
72 Gursoy-Ozdemir Y, Qiu J, Matsuoka N, et al. Cortical spreading depression activates and upregulates MMP-9. J Clin Invest. 2004; 113: 1447-1455.
73 Shah BH, Farshori MP, Jambusaria A, et al. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem. 2003; 278: 19118-19126.
74 Shah BH, Farshori MP, Catt KJ. Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor. J Biol Chem. 2004; 279: 414 -420.
75 Shah BH, Neithardt A, Chu DB, et al. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J Cell Physiol. 2006; 206: 47-57.
76 Piiper A, Dikic I, Lutz MP, et al. Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells. J Biol Chem. 2002; 277: 43623-43630.
77 Baron C, Benes C, Van Tan H, et al. Potassium chloride pulse enhances mitogen-activated protein kinase activity in rat hippocampal slices. J Neurochem. 1996. 66:1005-1010.
78 Corvol JC, Valjent E, Toutant M, et al. Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices. J Biol Chem. 2005; 280: 660-668.
79 Vanning T, Christopherson P, Schousboe A, et al. Pharmacological characterisation of voltage-sensitive calcium channels and neurotransmitter release from mouse cerebellar granule cells in culture. J Neurosci Res. 1997; 48: 43-52.
80 English JD, Sweatt JD. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem. 1996; 271: 24329-24332.
81 Yuan LL, Adams JP, Swank M, et al. Protein kinase modulation of dendriticK+ channels in hippocampus involves a mitogenactivated protein kinase pathway. J Neurosci. 1996; 22: 4860-4868.
82 Balazs R. Trophic effect of glutamate. Curr Top Med Chem. 2006; 6: 961-968.
83 Atkins CM, Selcher JC, Petraitis JJ, et al. The MAPK cascade is required for mammalian associative learning. Nat Neurosci. 1998; 1: 602-609.
84 Winder DG, Martin KC, Muzzio IA, et al. ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors. Neuron. 1999; 24: 715-726.
85 Dudek SM, Fields RD. Mitogen-activated protein kinase/ extracellular signal-regulated kinase activation in somatodendritic compartments: roles of action potentials, frequency, and mode of calcium entry. J Neurosci. 2001; 21: 1-5.
86 Huber KM, Mauk MD, Thompson C, et al. A critical period of protein kinase activity after tetanic stimulation is required for the induction of long-term potentiation. Learn Mem. 1995; 2: 81-100.
87 D'Angelo E, Rossi P, Gall D, et al. Long-term potentiation of synaptic transmission at the mossy fiber-granule cell relay of cerebellum. Prog Brain Res. 2005; 148: 69-80.
88 Nieus T, Sola E, Mapelli J, et al. LTP regulates burst initiation and frequency at mossy fiber-granule cell synapses of rat cerebellum: experimental observations and theoretical predictions. J Neurophysiol. 2006; 95: 686-699.
89 Mapelli J, D'Angelo E. The spatial organization of longterm synaptic plasticity at the input stage of cerebellum. J Neurosci. 2007; 27: 1285-1296.
90 Gall D, Prestori F, Sola E, et al. Intracellular calcium regulation by burst discharge determines bi-directional long-term synaptic plasticity at the cerebellum input stage. J Neurosci. 2007; 25: 4813-4822.
1 Pandiella A, Massague J. Cleavage of the membrane precursor for transforming growth factor alpha is a regulated process. Proc Natl Acad Sci U S A. 1991; 88:1726-1730
2 Harris DA, Walter NG. Probing RNA structure and metal-binding sites using terbium (III) footprinting. Curr Protoc Nucleic Acid Chem. 2003; Chapter 6: Unit 6.8.
3 Birecree E, King LE Jr, Nanney LB . Epidermal growth factor and its receptor in the developing human nervous system. Brain Res Dev Brain Res. 1991; 60: 145-154
4 Nakagawa T, Sasahara M, Hayase Y, et al. Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat. Dev. Brain Res. 1998; 108: 263-272.
5 Falk A, Frisen J . Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res. 2002; 69: 757-762.
6 Carrasco E, Blum M, Weickert CS, et al. Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone. Brain Res Dev Brain Res. 2003; 140: 1-13.
7 Lu YC, Qi JG, Wang TH, et al. Distribution of epidermal growth factor in central nervous system of adult rhesus monkey. Sichuan Da Xue Xue Bao Yi Xue Ban. 2005; 36: 618-621.
8 Higashiyama S, Abraham JA, Miller J, et al. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991; 251: 936-939.
9 Jin K, Mao X, Sun Y, Xie L, et al. Heparine-binding epidermal growth factor-like growth factor: hypoxia-inducible expression in vitro and stimulation of neurogenesis in vitro and in vivo. J Neurosci. 2002; 22: 5365-5373.
10 Kornblum H, Zurcher S, Werb Z, et al. Multiple trophic actions of heparin-binding epidermal growth factor HB-EGF in the central nervous system. Eur. J. Neurosci. 1999; 11: 3236-3246.
11 Opanashuk L, Mark R, Porter J, et al. Heparin-binding epidermal growth factor-like growth factor in hippocampus: modulation of expression by seizures and anti-excitotoxic action. J Neurosci 1999; 19: 133-146.
12 Nakagawa T, Sasahara M, Hayase Y, et al. Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat. Dev Brain Res 1998; 108:263-272.
13 Grewal, J, Luttrell L, Raymond J . G protein-coupled receptors desensitize and down-regulate epidermal growth factor recptor in renal mesangial cells. J Biol Chem. 2001; 276: 27335-27344.
14 Tanaka N, Sasahara M, Ohno M, et al. Heparin-binding epidermal growth factor-like growth factor mRNA expression in neonatal rat brain with hypoxic/ischemic injury. Brain Res. 1999; 827: 130-138.
15 Gu L, Li B, Yang X, Hu X, et al. Depolarization-induced, glutamate Receptor-mediated and Transactivation-dependent Extracellular-signal Regulated Kinase Phosphorylation in Cultured Cerebellar Granule Neurons. Neurosci. 2007; 147: 342-353
16 Li B, Du T, Li H, et al. Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons.Br J Pharmacol. 2008; 154: 191-203.
17 Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer. 2001; 8: 11-31.
18 Krainock R, Murphy S . Heregulin upregulates the expression of nitric oxide synthase NOS-1 in rat cerebellar granule neurons via the ErbB4 receptor. J Neurochem. 2001; 76: 312-315.
19 Lezama R, Ortega A, Ordaz B, et al. Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons. J Neurochem. 2005; 93: 1189-1198.
20 Pinkas-Kramarski, R, Eilam, R, Alroy, I, et al. Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation. Oncogene. 1997; 15:2803-2815.
21 Huang, Y, Wang, Q, Xiong W.C, et al. Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95. J Biol Chem. 2001; 276: 19318-19326.
22 Beerli R, Hynes NE . Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J Biol Chem. 1996; 15 : 6071-6076.
23 Jones F, Golding J, Gassmann M. ErbB4 signaling during breast and neural development: novel genetic models reveal unique ErbB4 activities.Cell Cycle. 2003; 2: 555-559.
24 Law A, Shannon Weickert C, Hyde TM, et al. Neuregulin-1 NRG-1 mRNA and protein in the adult human brain. Neurosci. 2004; 127:125-136.
25 Rio C, Rieff HI, Qi P, et al. Neuregulin and ErbB receptors play a critical role in neuronal migration.Neuron. 1997; 19: 39-50.
26 Daub H, Wallasch C, Lankenau A, et al. Signal characteristics of G protein transactivated EGF receptor. EMBO J. 1997; 16: 7032-7044.
27 Peavy R, Chang M, Sanders B, et al. Metabotropic glutamate receptor 5-induced phosphorylation of extracellular signal-regulated kinase in astrocytes depends on transactivation of the epidermal growth factor receptor. J Neurosci. 2001; 21: 9619-9628.
28 Kalmes A, Vesti B, Daum G, et al. Heparin blockage of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor EGF receptor transactivation by heparin-binding EGF-like growth factors. Circ Res. 2000; 87: 92-98.
29 Saito S, Frank G, Motley E, et al. Metalloprotease inhibitor blocks angiotensin -induced migration through inhibition of epidermal growth factor receptor transactivation. Biochem Biophys Res Commun. 2002; 294: 1023-1029.
30 Goishi K, Higashiyama S, Klagsbrun M, et al. Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity. Mol Biol Cell. 1995; 6: 967-980.
31 Umata T, Hirata M, Takahashi T, et al. A dual signaling cascade that regulates the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. J. Biol. Chem. 2001; 276: 30475-30482
32 Izumi Y, Hirata M, Hasuwa H,A metalloprotease-disintegrin, MDC9/meltrin-gamma/ ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J . 1998; 17: 7260-7272.
33 Dethlefsen S, Raab, G, Moses, et al. Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. J Cell Biochem. 1998; 69:143-153.
34 Zwick E, Daub H, Aoki N, et al. Critical role of calcium- dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem 1997; 272: 24767-24770.
35 Tebar F, Llado A, Enrich C, et al. Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC. FEBS Lett. 2002; 517: 206-210.
36 Cussac D, Schaak S, Denis C, et al. Alpha 2B-adrenergic receptor activates MAPK via a pathway involving arachidonic acid metabolism, matrix metalloproteinases, and epidermal growth factor receptor transactivation. J Biol Chem. 2002; 277:19882-198828.
37 Cussac D, Schaak S, Gales C, et al. Flordellis C, Denis C, Paris H.alpha (2B) -Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells. Am J Physiol Renal Physiol. 2002; 282: 943-952.
38 Pierce K, Tohgo A, Ahn S, et al. Epidermal growth factor EGF receptor-dependent ERK activation by G protein-coupled receptors. J Biol Chem. 2001; 276: 23155-23160.
39 Bruno V, Battaglia G, Casabona G, et al. Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta. J. Neurosci. 1998; 18: 9594-9600.
40 Zelenaia O, Schlag B, Gochenauer G, et al. Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. Mol. Pharmacol. 2000; 57: 667-678.
41 Xue L, Murray J, Tolkovsky A. The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J Biol Chem. 2000; 275: 8817-8824.
42 Shah BH, Farshori M, Jambusaria A, et al. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation.J Biol Chem. 2003; 278: 19118-191126.
43 Shah BH, Farshori MP, Catt KJ. Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor. J Biol Chem. 2004; 279: 414-420.
44 Shah BH, Neithardt A, Chu DB, et al. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J Cell Physiol. 2006; 206: 47-57.
45 Piiper A, Dikic I, Lutz MP, et al. Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells. J Biol Chem. 2002; 277: 43623-43630.
46 Shah BH, Farshori MP, Jambusaria A, et al. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERKl/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem. 2003; 278: 19118-19126.
47 Boonstra J, De Laat SW, Ponec M. Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes. Exp Cell Res. 1985; 161: 421-433.
48 Traverse, S., Seedorf, K., Paterson, H., et al. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol. 1994; 4; 694-701.
49 49 Soltoff SP, Cantley LC. 120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC 12 and other cells. J Biol Chem. 1996; 271: 563-567.
50 Klesse LJ, Meyers KA, Marshall CJ, et al. Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells. Oncogene. 1999; 18: 2055-2068.
51 Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995; 267: 2003-2006.
52 Jackson, T.R., Blader, I.J., Hammonds-Odie, L.P., et al. Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase. J Cell. Sci. 1996; 109: 289-300.
53 Kimura, K., Hattori, S., Kabuyama, Y., et al. Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 1994; 269: 18961-18967.
54 Posern, G., Saffrich, R., Ansorge, W., et al. Rapid lamellipodia formation in nerve growth factor-stimulated PC12 cells is dependent on Rac and PI3K activity. J Cell Physiol. 2000; 183; 416-424.
55 Yasui H, Katoh H., Yamaguchi Y, et al. Differential responses to nerve growth factor and epidermal growth factor in neurite outgrowth of PC12 cells are determined by Racl activation systems. J Biol Chem. 2001; 276: 15298-15305.
56 Corbit KC, Soh JW, Yoshida K, et al. Different protein kinase C isoforms determine growth factor specificity in neuronal cells.Mol Cell Biol. 2000; 20: 5392-5403.
57 Lev, S, Moreno H, Martinez R, et al. Protein tyrosine kinase PYK2 involved in Ca (2+) -induced regulation of ion channel and MAP kinase functions. Nature. 1995; 376: 737-745.
58 Coogan AN, Piggins HD. Circadian and photic regulation of phosphorylation of ERK1/2 and Elk-1 in the suprachiasmatic nuclei of the Syrian hamster. J Neurosci. 2003; 23: 3085-3093.
59 Kuroda KO, Meaney MJ, Uetani N, et al. ERK-FosB signaling in dorsal MPOA neurons plays a major role in the initiation of parental behavior in mice. Mol Cell Neurosci. 2007; 36: 121-131.
60 English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 1997; 272: 19103-19106.
61 Di Segni A, Farin K, Pinkas-Kramarski R. ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells: involvement of PI3K and Erk signaling. J Mol Neurosci. 2006; 29: 257-267.
62 Jin K, Sun Y, Xie L, et al. Post-ischemic administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) reduces infarct size and modifies neurogenesis after focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 2004; 24: 399-408.
63 Michalsky M, Kuhn A, Mehta V, et al. Heparin-binding EGF-like growth factor decreases apotosis in intestinal epithelial cells in vitro. J Pediatr Surg. 2001; 36: 1130-1135.
64 Lara-Marquez M, Mehta V, Michalsky M, et al. Heparin-binding EGF-like growth factor down regulates proinflammatory cytokin-induced nitric oxide and inducible nitric oxide synthase production in intestinal epithelial cells. Nitric Oxide. 2005; 6: 142-152.
65 Bryson, J, Coy P, Gottlob K, et al. Increased hexokinase activity, or either ectopic or endogenous origin, protects renal epithelial cells against acute oxidant-induced cell death. J Biol Chem. 2002; 277: 11392-11400.
66 Zhu Y, Yang G, Ahlemeyer B, et al. Transforming growth factor-beta 1 increases bad phosphorylation and protects neurons against damage. J Neurosci. 2002; 22: 3898-3909.
67 Han B, Holtzman D . BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci. 2000; 20: 5775-5781.
68 Lenhard T, Schober A, Suter-Crazzolara C, et al. Fibroblast growth factor-2 requires glial-cell-line-derived neurotrophic factor for exerting its neuroprotective actions on glutamate-lesioned hippocampal neurons. Mol Cell Neurosci. 2002; 20: 181-197.