Regulation of Metabotropic Glutamate Receptor Signaling

代谢型谷氨酸受体信号传导的调节

• 批准号: 8557055
• 负责人: Katherine Roche
• 金额: $ 72.12万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557055
• 关键词: AMPA Receptors; Affect; Binding; Birth; Brain; C-terminal; Calcium; Calcium Oscillations; Calcium Signaling; Data; Development; Disease; Endocytosis; G-Protein-Coupled Receptors; Glutamates; Goals; Growth; Hippocampus (Brain); Hyperpigmentation; Immunoblotting; Leucine; Link; Malignant Neoplasms; Mediating; Metabotropic Glutamate Receptors; Molecular; Mus; Mutation; Neurons; PKC Phosphorylation Site; Penetrance; Phenotype; Phospholipase C; Phosphorylation; Phosphorylation Site; Prosencephalon; Proteins; Receptor Signaling; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Signal Transduction; Signaling Molecule; Site; Surface; Synapses; Synaptic plasticity; Tail; Therapeutic Studies; Tissues; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; beta-2 Adrenergic Receptors; desensitization; early onset; external ear auricle; in vivo; interest; melanocyte; melanoma; member; neurotransmission; neurotransmitter release; postsynaptic; promoter; protein protein interaction; receptor; receptor binding; receptor coupling; response; tool; trafficking; tumor

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 severephenotype, make these mouse lines unique and a potentially powerful tool for therapeutic studies.

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、Ca2+ 信号传导和受体脱敏。 I 组 mGluR（mGluR1 和 mGluR5）主要是突触后 mGluR，它们与磷脂酶 C 偶联，释放细胞内 Ca2+，并激活多种细胞内信号分子。 mGluR5 的 PKC 磷酸化影响 Ca2+ 信号传导和受体脱敏。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内。 Ser839 是一个磷酸化位点，决定响应 mGluR5 激活的细胞内钙振荡的调节。我们还表明，mGluR5 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受体和 PKC 激活而上调。此外，S901 磷酸化会抑制 mGluR5 与 CaM 的结合，从而降低 mGluR5 表面表达。此外，阻断 S901 上 mGluR5 的 PKC 磷酸化可通过延长 Ca2+ 振荡来显着影响 mGluR5 信号传导。因此，我们的数据表明 mGluR5 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传导。 虽然 mGluR1 和 mGluR5 都与 CaM 相互作用已被接受，但我们现在已经证明 CaM 特异性结合 mGluR5 而不是 mGluR1。我们已经确定了 mGluR5 (L896) 中 CaM 结合所需的一个关键残基。在 mGluR1 中，类似残基 V909 突变为亮氨酸足以赋予 CaM 与 mGluR1 的结合。为了研究 CaM 结合的功能影响，我们检测了海马神经元中 mGluR1 和 mGluR5 的表面表达。 mGluR1 (V909L) 中赋予 CaM 结合的突变显着增加 mGluR1 表面表达，而 mGluR5 中破坏 CaM 结合的类似突变 (L896V) 则降低 mGluR5 表面表达。此外，改变 CaM 结合的关键残基可调节 mGluR 内化。此外，我们发现 CaM 与 I 组 mGluR 结合可增强 mGluR 介导的 AMPA 受体内吞作用。最后，我们发现 I 组 mGluR 引起的钙反应受到这些突变的调节，从而调节 CaM 结合。我们的研究结果阐明了专门影响 mGluR5 运输和信号传导的关键机制，并区分了 mGluR1 和 mGluR5 调节。 尽管许多兴趣和研究都集中在中枢神经系统中正常 mGluR 信号传导的作用，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 激活的钙信号传导，我们使用 Thy1 启动子驱动前脑中的表达，生成了 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，我们在黑色素细胞特异性启动子 TRP1 下生成了 mGluR5 转基因系。大多数创始人表现出早期发病的严重表型。对于大多数 mGluR5 转基因阳性小鼠，早在出生后 3-5 天就可以检测到耳廓和尾部色素沉着过度。从患有黑色素瘤的创始人产生的 TRP1-mGluR5 系的后代中，外显率为 100%。通过 RT-PCR 和免疫印迹检测黑色素瘤样本中 mGluR5 的表达。我们评估了肿瘤样本中几种癌症相关蛋白的表达，并观察到 ​​ERK 磷酸化显着增加，表明 ERK 作为肿瘤中 mGluR5 信号传导的下游效应器。我们的研究结果表明，mGluR5 介导的谷氨酸信号传导可以在体内引发黑色素瘤。积极的生长和严重的表型使这些小鼠品系独一无二，并成为治疗研究的潜在强大工具。