Regulation of Metabotropic Glutamate Receptor Signaling

MGluRs是GPCRs，调节兴奋性神经传递、神经递质释放和突触可塑性。PKC调节mGluR功能的许多方面，包括蛋白质-蛋白质相互作用、钙信号转导和受体脱敏。I组mGluR(mGluR1和mGluR5)主要是突触后mGluR，与磷脂酶C偶联，释放细胞内钙离子，激活多种细胞内信号分子。MGluR5的PKC磷酸化影响钙信号转导和受体脱敏。我们已经确定了几个PKC位点，它们位于mGluR5 C-末端结构域的近三分之一处。一个名为Ser839的磷酸化位点决定了mGluR5激活后细胞内钙振荡的调节。我们还发现，mGluR5细胞内C末端的主要PKC磷酸化位点是S901，该残基的磷酸化水平随着受体和PKC的激活而上调。此外，S901的磷酸化抑制了mGluR5与CaM的结合，降低了mGluR5的表面表达。有趣的是，缺失的E3连接酶7(Siah)-1A与mGluR5结合，并与CaM结合竞争。在最近的研究中，我们证明了CaM在神经元中以一种磷酸化依赖的方式与Siah-1A竞争mGluR5结合。我们发现mGluR5 S901的磷酸化通过取代CaM而增强了Siah-1A的结合。为了阐明Siah-1A与mGluR5结合的分子基础，我们使用突变和生化技术来鉴定调节Siah-1A与mGluR5结合的关键残基。我们发现mGluR5与Siah-1A的结合是Siah-1A对mGluR5运输的影响所必需的。具体地说，我们发现Siah-1A结合降低了mGluR5的表面表达，增加了mGluR5的内体转运和溶酶体降解。 在其他研究中，我们研究了通过磷酸化对突触前受体mGluR7的调节。MGluR7定位于突触前活动区，在那里它作为一种自身受体抑制神经递质的释放。我们先前已经证明，mGluR7在Ser-862上的PKC磷酸化是通过调节CaM和与C激酶相互作用的蛋白(PICK1)的竞争性相互作用来控制mGluR7的结构性和活性依赖的表面表达的关键机制。由于受体的磷酸化和去磷酸化是通过蛋白激酶和磷酸酶的精确作用而紧密协调的，我们现在已经确定了磷酸酶在调节mGluR7受体表面表达的活性依赖或激动剂诱导的变化中的作用。我们发现丝氨酸/苏氨酸蛋白磷酸酶1(PP1)在mGluR7上丝氨酸/苏氨酸蛋白磷酸酶1(PP1)的结构性和激动剂诱导的Ser-862去磷酸化中起着至关重要的作用。用PP1抑制剂处理神经元后，Ser-862的磷酸化和mGluR7的表面表达都显著增加。此外，mGluR7a和mGluR7b的Ser-862磷酸化是PP1的靶标。有趣的是，激动剂诱导的mGluR7去磷酸化受PP1调节，而NMDA介导的活性诱导的去磷酸化不受PP1调节，这表明有多种信号通路影响受体的磷酸化和转运。 虽然大部分的兴趣和研究都集中在正常的mGluR信号在中枢神经系统中的作用上，但mGluR也在非神经元组织中表达，并与包括癌症在内的多种疾病有关。为了研究mGluR激活的神经元钙信号，我们用Thy1启动子驱动前脑表达的mGluR5转基因动物，其中一位创始人意外地患上了黑色素瘤。为了直接研究mGluR5在黑色素瘤形成中的作用，我们在黑素细胞特异性启动子TRP1下建立了mGluR5转基因株系。大多数创始人表现出严重的表型，起病早。大多数mGluR5转基因阳性小鼠在出生后3-5天就可以检测到羽片和尾巴的色素沉着。从患有黑色素瘤的创建者产生的TRP1-mGluR5系的后代中，有100%的外显率。RT-PCR和免疫印迹法检测到黑色素瘤组织中mGluR5的表达。我们评估了几个肿瘤相关蛋白在肿瘤样本中的表达，并观察到ERK的磷酸化显著增加，暗示ERK是肿瘤中mGluR5信号的下游效应因子。我们的研究结果表明，mGluR5介导的谷氨酸能信号转导可以在体内触发黑色素瘤。侵略性的生长和严重的表型，使这些小鼠系独一无二，并可能成为治疗研究的强大工具。我们目前的研究集中在定义mGluR5上的区域，这些区域是在转基因小鼠中引发黑色素瘤的关键。我们正在使用受体截断和嵌合体，研究细胞在体外的转化以及体内的肿瘤形成。

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. Interestingly, the E3 ligase seven in absentia homolog (Siah)-1A binds to mGluR5 and competes with CaM binding. In recent studies, we demonstrated that CaM competes with Siah-1A for mGluR5 binding in a phosphorylation-dependent manner in neurons. We showed that phosphorylation of mGluR5 S901 enhances Siah-1A binding by displacing CaM. To elucidate the molecular basis for Siah-1A binding, we used mutagenesis and biochemical techniques to identify critical residues regulating Siah-1A binding to mGluR5. We found that mGluR5 binding to Siah-1A is essential for the Siah-1A effects on mGluR5 trafficking. Specifically, we found that Siah-1A binding decreases mGluR5 surface expression and increases endosomal trafficking and lysosomal degradation of mGluR5. In other studies, we have investigated the regulation of the presynaptic receptor, mGluR7, by phosphorylation. mGluR7 is localized to the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. We have previously shown that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of CaM and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, we have now characterized the role of phosphatases in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. We found that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. 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 severephenotype, make these mouse lines unique and a potentially powerful tool for therapeutic studies. Our current studies are focused on defining the domains on mGluR5 that are essential for triggering melanoma in the transgenic mice. We are using receptor truncations and chimeras and studying cell transformation in vitro as well as tumor formation in vivo.