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，包括蛋白相互作用，Ca2+信号传导和受体脱敏。I组mGluRs （mGluR1和mGluR5）主要是突触后mGluRs，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。PKC磷酸化mGluR5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。一个磷酸化位点Ser839决定了响应于mGluR5激活的细胞内钙振荡的调节。我们还发现mGluR5细胞内C端PKC的主要磷酸化位点是S901，该残基的磷酸化在受体和PKC激活的情况下均上调。此外，S901磷酸化抑制mGluR5与CaM结合，降低mGluR5表面表达。有趣的是，E3连接酶7缺失同源物(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在神经元中以磷酸化依赖的方式与Siah-1A竞争mGluR5的结合。我们发现mglur5s901的磷酸化通过取代CaM增强了Siah-1A的结合。为了阐明Siah-1A结合的分子基础，我们使用诱变和生化技术鉴定了调节Siah-1A与mGluR5结合的关键残基。我们发现mGluR5与Siah-1A的结合对于Siah-1A对mGluR5运输的影响至关重要。具体来说，我们发现Siah-1A结合降低了mGluR5表面表达，增加了mGluR5的内体运输和溶酶体降解。

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.