Mechanisms associated with activation of intracellular metabotropic glutamate receptor, mGluR5

The group 1 metabotropic glutamate receptor, mGluR5, is found on the cell surface as well as on intracellular membranes where it can mediate both overlapping and unique signaling effects. Previously we have shown that glutamate activates intracellular mGluR5 by entry through sodium-dependent transporters and/or cystine glutamate exchangers. Calibrated antibody labelling suggests that the glutamate concentration within neurons is quite high (~10 mM) raising the question as to whether intracellular mGluR5 is maximally activated at all times or whether a different ligand might be responsible for receptor activation. To address this issue, we used cellular, optical and molecular techniques to show that intracellular glutamate is largely sequestered in mitochondria; that the glutamate concentration necessary to activate intracellular mGluR5 is about ten-fold higher than what is necessary to activate cell surface mGluR5; and uncaging caged glutamate within neurons can directly activate the receptor. Thus these studies further the concept that glutamate itself serves as the ligand for intracellular mGluR5

Sequences within the C terminus of the metabotropic glutamate receptor 5 (mGluR5) are responsible for inner nuclear membrane localization

Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca(2+) changes in the nucleoplasm by coupling with G(q/11). Here, we identified a region within the C-terminal domain (amino acids 852–876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca(2+) in situ

Striatal mGlu5-mediated synaptic plasticity is independently regulated by location-specific receptor pools and divergent signaling pathways

Metabotropic glutamate receptor 5 (mGl

Intracellular mGluR5 plays a critical role in neuropathic pain

Spinal mGluR5 is a key mediator of neuroplasticity underlying persistent pain. Although brain mGluR5 is localized on cell surface and intracellular membranes, neither the presence nor physiological role of spinal intracellular mGluR5 is established. Here we show that in spinal dorsal horn neurons >80% of mGluR5 is intracellular, of which ∼60% is located on nuclear membranes, where activation leads to sustained Ca(2+) responses. Nerve injury inducing nociceptive hypersensitivity also increases the expression of nuclear mGluR5 and receptor-mediated phosphorylated-ERK1/2, Arc/Arg3.1 and c-fos. Spinal blockade of intracellular mGluR5 reduces neuropathic pain behaviours and signalling molecules, whereas blockade of cell-surface mGluR5 has little effect. Decreasing intracellular glutamate via blocking EAAT-3, mimics the effects of intracellular mGluR5 antagonism. These findings show a direct link between an intracellular GPCR and behavioural expression in vivo. Blockade of intracellular mGluR5 represents a new strategy for the development of effective therapies for persistent pain

Sequences Located within the N-Terminus of the PD-Linked LRRK2 Lead to Increased Aggregation and Attenuation of 6-Hydroxydopamine-Induced Cell Death

<div><p>Clinical symptoms of Parkinson's disease (PD) arise from the loss of substantia nigra neurons resulting in bradykinesia, rigidity, and tremor. Intracellular protein aggregates are a pathological hallmark of PD, but whether aggregates contribute to disease progression or represent a protective mechanism remains unknown. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to PD in both familial cases and idiopathic cases and aggregates of the LRRK2 protein are present in postmortem PD brain samples. To determine whether LRRK2 contains a region of protein responsible for self-aggregation, two independent, bioinformatic algorithms were used to identify an N-terminal amino acid sequence as being aggregation-prone. Cells subsequently transfected with a construct containing this domain were found to have significantly increased protein aggregation compared to wild type protein or a construct containing only the last half of the molecule. Finally, in support of the hypothesis that aggregates represent a self-protection strategy, aggregated N-terminal LRRK2 constructs significantly attenuated cell death induced by the PD-mimetic, 6-hydroxydopamine (6-OHDA).</p> </div

Predicted LRRK2 aggregation domains.

<p>(<b>A</b>) The PASTA algorithm by Trovato <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045149#pone.0045149-Trovato2" target="_blank">[19]</a> predicts an aggregation domain at 210–310 amino acid residues in the LRRK2 protein (NCBI accession number NP_940980). (<b>B</b>) An independent aggregation algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045149#pone.0045149-Tartaglia2" target="_blank">[21]</a> predicts two aggregation domains, one peak between amino acid residues (250 to 299) overlapping with the N-terminus region predicted by PASTA (A) and the other at amino acid residues 2050–2099. Tick marks in B represent sliding window of 50 amino acids.</p

Increased N-term-LRRK2 aggregation in mesencephalic neurons.

<p>(<b>A</b>) Representative images showing N-term-LRRK2 cells with increased high intensity aggregates (arrows) in the cell body and neurites compared to WT-, C-term-, or N-del-LRRK2 constructs. Inset of the cell body is enlarged (bottom) to illustrate aggregates (arrows). Aggregates were quantitated utilizing number of granules/site (<b>B</b>), total granule area/site (<b>C</b>), and average granule intensity per site (<b>D</b>). Data shown are average of 3 independent experiments with more than 6 dishes/construct and 64 images/dish. (** P<0.01, ***P<0.001, one-way ANOVA with Bonferroni <i>post hoc</i> test). (<b>E</b>) Representative images showing the colocalization of EGFP-LRRK2 with MAP2 or TH immunostaining. Increased aggregates were observed in N-term-LRRK2 neurons including dopaminergic neurons. (<b>F</b>) Quantitation of numbers of LRRK2/MAP2 positive neurons with aggregates. Bars represent means of three experiments ± S.E.M. with multiple dishes and a total of about 600 MAP2 positive neurons analyzed. (*** P<0.001, one-way ANOVA with Bonferroni <i>post hoc</i> test). (<b>G</b>) Quantitation of numbers of LRRK2/TH-positives neurons with aggregates. Bars represent mean values from each of 4 experiments ± S.E.M. A total of about 150 dopaminergic neurons were analyzed (*** P<0.001, one-way ANOVA with Bonferroni <i>post hoc</i> test).</p