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。此外，阻断PKC在S901上的mGluR5磷酸化，通过延长Ca2+振荡显著影响mGluR5信号传导。因此，我们的数据表明mGluR5激活触发S901的磷酸化，从而直接连接PKC磷酸化、CaM结合、受体运输和下游信号传导。

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.