Regulation of Metabotropic Glutamate Receptor Signaling

mGluR是调节兴奋性神经传递、神经递质释放和突触可塑性的GPCR。PKC调节mGluR功能的许多方面，包括蛋白质-蛋白质相互作用、Ca 2+信号传导和受体脱敏。I组mGluR（mGluR 1和mGluR 5）主要是突触后mGluR，与磷脂酶C偶联，释放细胞内Ca 2+，激活多种细胞内信号分子。mGluR 5的PKC磷酸化影响Ca 2+信号传导和受体脱敏。我们已经确定了几个PKC网站，这是位于近三分之一的mGluR 5的C-末端结构域。一个磷酸化位点，Ser 839，决定响应mGluR 5激活的细胞内钙振荡的调节。我们还表明，mGluR 5的胞内C末端的主要PKC磷酸化位点是S901，并且该残基的磷酸化响应于受体和PKC激活而上调。此外，S901磷酸化抑制mGluR 5与CaM结合，降低mGluR 5表面表达。此外，阻断S901上mGluR 5的PKC磷酸化通过延长Ca 2+振荡显著影响mGluR 5信号传导。因此，我们的数据表明，mGluR 5激活触发S901磷酸化，从而直接连接PKC磷酸化，CaM结合，受体运输和下游信号。 虽然已经接受mGluR 1和mGluR 5都与CaM相互作用，但我们现在已经证明CaM特异性结合mGluR 5而不是mGluR 1。我们已经确定了一个单一的关键残基mGluR 5（L 896），这是钙调素结合所需的。在mGluR 1中，类似残基V909突变为亮氨酸足以使CaM与mGluR 1结合。为了研究钙调素结合的功能效应，我们研究了海马神经元中mGluR 1和mGluR 5的表面表达。赋予CaM结合的mGluR 1突变（V909 L）显著增加mGluR 1表面表达，而破坏CaM结合的mGluR 5类似突变（L 896 V）降低mGluR 5表面表达。此外，改变钙调素结合的关键残基调节mGluR内化。此外，我们发现，mGluR介导的AMPA受体的内吞作用增强钙调素结合组I mGluRs。最后，我们表明，由I组mGluRs引起的钙反应调制这些突变，调节钙调素结合。我们的研究结果阐明了一个关键的机制，专门影响mGluR 5的运输和信号，并区分mGluR 1和mGluR 5的调节。 尽管许多兴趣和研究集中在CNS中正常mGluR信号传导的作用上，但mGluR也在非神经元组织中表达，并且与包括癌症在内的多种疾病有关。为了研究神经元中mGluR激活的钙信号，我们使用Thy 1启动子来驱动前脑中的表达，产生了mGluR 5转基因动物，其中一个创始人意外地发展了黑色素瘤。为了直接研究mGluR 5在黑色素瘤形成中的作用，我们在黑色素细胞特异性启动子TRP 1下产生了mGluR 5转基因株系。大多数创始人表现出早期发病的严重表型。大多数mGluR 5转基因阳性小鼠在出生后3-5天就可以检测到鳍和尾部的色素沉着。在由产生黑色素瘤的创始人产生的TRP 1-mGluR 5系的后代中有100%的突变率。通过RT-PCR和免疫印迹检测黑素瘤样品中mGluR 5的表达。我们评估了肿瘤样品中几种癌症相关蛋白的表达，并观察到ERK磷酸化的显著增加，暗示ERK是肿瘤中mGluR 5信号传导的下游效应子。我们的研究结果表明，mGluR 5介导的谷氨酸能信号可以触发体内黑色素瘤。侵袭性生长和严重的表型，使这些小鼠品系独特，并成为治疗研究的潜在强大工具。

The mGluRs are GPCRs that modulate excitatory neurotransmission, neurotransmitter release, and synaptic plasticity. PKC regulates many aspects of mGluR function, including protein-protein interactions, Ca2+ signaling, and receptor desensitization. The group I mGluRs (mGluR1 and mGluR5) are predominantly postsynaptic mGluRs that are coupled to phospholipase C, release of intracellular Ca2+, and activation of a variety of intracellular signaling molecules. PKC phosphorylation of mGluR5 affects Ca2+ signaling and receptor desensitization. We have identified several PKC sites, which are located within the proximal one-third of the mGluR5 C-terminal domain. One phosphorylation site, Ser839, determines the regulation of intracellular calcium oscillations in response to mGluR5 activation. We have also shown that the major PKC phosphorylation site on the intracellular C terminus of mGluR5 is S901, and phosphorylation of this residue is up-regulated in response to both receptor and PKC activation. In addition, S901 phosphorylation inhibits mGluR5 binding to CaM, decreasing mGluR5 surface expression. Furthermore, blocking PKC phosphorylation of mGluR5 on S901 dramatically affects mGluR5 signaling by prolonging Ca2+ oscillations. Thus, our data demonstrate that mGluR5 activation triggers phosphorylation of S901, thereby directly linking PKC phosphorylation, CaM binding, receptor trafficking, and downstream signaling. Although it has been accepted that both mGluR1 and mGluR5 interact with CaM, we have now shown that CaM specifically binds to mGluR5 and not mGluR1. We have identified a single critical residue in mGluR5 (L896) that is required for CaM binding. In mGluR1, mutation of the analogous residue, V909, to leucine is sufficient to confer CaM binding to mGluR1. To investigate the functional effects of CaM binding, we examined the surface expression of mGluR1 and mGluR5 in hippocampal neurons. The mutation in mGluR1 (V909L) that confers CaM binding dramatically increases mGluR1 surface expression, whereas the analogous mutation in mGluR5 that disrupts CaM binding (L896V) decreases mGluR5 surface expression. In addition, the critical residue that alters CaM binding regulates mGluR internalization. Furthermore, we find that mGluR-mediated AMPA receptor endocytosis is enhanced by CaM binding to group I mGluRs. Finally, we show that calcium responses evoked by group I mGluRs are modulated by these mutations, which regulate CaM binding. Our findings elucidate a critical mechanism that specifically affects mGluR5 trafficking and signaling, and distinguishes mGluR1 and mGluR5 regulation. Although much of the interest and research has focused on the role of normal mGluR signaling in the CNS, mGluRs are also expressed in non-neuronal tissues and have been implicated in a variety of diseases including cancer. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, TRP1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3-5 days after birth for most of mGluR5 transgene positive mice. There was 100% penetrance in the progeny from the TRP1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by both RT-PCR and immunoblotting. We evaluated the expression of several cancer related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5 mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype, make these mouse lines unique and a potentially powerful tool for therapeutic studies.