Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A‐dependent manner in non‐neuronal and neuronal cell lines

Trafficking of G protein‐coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein‐coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non‐neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand‐mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand‐mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A‐dependent manner.

     

Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.     

Homer 1b regulates the trafficking of group I metabotropic glutamate receptors.

The molecular basis for glutamate receptor trafficking to the plasma membrane is not understood. In the present study, we demonstrate that Homer 1b (H1b), a constitutively expressed splice form of the immediate early gene product Homer (now termed Homer 1a) regulates the trafficking and surface expression of group I metabotropic glutamate receptors. H1b inhibits surface expression of the metabotropic glutamate receptor mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum (ER). In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to the plasma membrane. In addition, point mutations that disrupt mGluR5 binding to H1b eliminate ER retention of mGluR5, demonstrating that H1b affects metabotropic receptor localization via a direct protein-protein interaction. Electron microscopic analysis reveals that the group I metabotropic receptor mGluR1alpha is significantly enriched in the ER of Purkinje cells, suggesting that a similar mechanism may exist in vivo. Because H1b is found in dendritic spines of neurons, local retention of metabotropic receptors within dendritic ER provides a potential mechanism for regulating synapse-specific expression of group I metabotropic glutamate receptors.     

Cell-type-specific pathways of neurotensin endocytosis

The neurotensin receptor subtype 1 (NTS1) is a G-protein-coupled receptor (GPCR) mediating a large number of central and peripheral effects of neurotensin. Upon stimulation, NTS1 is rapidly internalized and targeted to lysosomes. This process depends on the interaction of the phosphorylated receptor with β–arrestin. Little is known about other accessory endocytic proteins potentially involved. Here, we investigated the involvement of dynamin, amphiphysin, and intersectin in the internalization of NTS1 receptor-ligand complexes in transfected COS-7 and HEK 293 cells, by using the transferrin receptor as an internal control for the constitutive endocytic pathway. We found that NTS1 endocytosis was not only arrestin–dependent, but also dynamin–dependent in both COS-7 and HEK 293 cells, whereas internalization of the transferrin receptor was independent of arrestin but required dynamin. Overexpression of the SH3 domain of amphiphysin II had no effect on receptor internalization in either cell type. By contrast, overexpression of full-length intersectin or of its SH3 domain (but not of its EH domain) inhibited NTS1 internalization in COS-7 but not in HEK 293 cells. This difference between COS-7 and HEK 293 cells was not attributable to differences in endogenous intersectin levels between the two cell lines. Indeed, the same constructs inhibited transferrin endocytosis equally well in COS-7 and HEK 293 cells. However, immunogold electron microscopy revealed that internalized NTS1 receptors were associated with clathrin-coated pits in COS-7 cells but with smooth vesicles in HEK 293 cells, suggesting that NTS1 internalization proceeds via different endocytic pathways in these two cell types. This work was supported by grants to A.B. from CIHR and FRSQ.     

Co-stimulation of mGluR5 and N-methyl-D-aspartate receptors is required for potentiation of excitatory synaptic transmission in hippocampal neurons

In the central nervous system, excitatory synaptic transmission is mediated by the neurotransmitter glutamate and its receptors. Interestingly, stimulation of group I metabotropic glutamate receptors (mGluRs) can either enhance or depress synaptic transmission at CA1 hippocampal synapses. Here we report that co-activation of mGluR5, a member of the group I mGluR family, and N-methyl-d-aspartate receptors (NMDARs) potentiates NMDAR currents and induces a long lasting enhancement of excitatory synaptic transmission in primary cultured hippocampal neurons. Unexpectedly, activation of mGluR5 alone fails to enhance evoked NMDAR currents and synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) AMPAR currents. The observed potentiation requires an mGluR5-induced, inositol 1,4,5-trisphosphate receptor-mediated mobilization of intracellular Ca2+, which acts in concert with a protein kinase C, calcium-activated tyrosine kinase cascade to induce a long lasting enhancement of NMDAR and AMPAR currents.     

The C-terminus of the metabotropic glutamate receptor 1b regulates dimerization of the receptor

The Group C G protein-coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABA_B receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo- or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C-termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C-terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co-expression of mGluR1a and mGluR1b in COS-7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C-terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a.     

Endocytosis of adiponectin receptor 1 through a clathrin- and Rab5-dependent pathway

In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogenesis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Eps15 mutants or depleting K(+) trapped AdipoR1 at the plasma membrane, and K(+) depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoR1 and adiponectin is clathrin-dependent. Depletion of K(+) and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might downregulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoR1 is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.     

Metabotropic glutamate receptor/phospholipase C pathway is increased in rat brain at the end of pregnancy

Wistar pregnant rats were sacrificed at the end of pregnancy and the status of metabotropic glutamate receptors/phospholipase C (mGluR/PLC) pathway was studied in brain from pregnant and non-pregnant female rats. Pregnancy causes a significant increase in metabotropic glutamate receptors number, determined by radioligand binding assay, without significant changes on receptor affinity. Similar increase in mGluR(1) type was obtained by immunoblotting assay using specific anti-mGluR(1) antibody. However, no significant differences were observed in mGluR(5) type, suggesting that the increase detected by radioligand assays could be due to mGluR(1) up-regulation. On the other hand, a significant increase in the alpha subunit of G(q) protein was also detected in pregnant rats by immunoblotting assays. Real-time PCR experiments revealed a significant increase in gene expression of metabotropic glutamate receptors and G(q) proteins. Neither protein level nor gene expression of phospholipase C beta(1) isoform was altered in pregnant rats. However, an increase in basal and agonist-stimulated phospholipase C activity was observed in membranes from pregnant rats. These results suggest that gestational period causes the up-regulation of both metabotropic glutamate receptors and coupled G(q)-protein and, in turn, an increase in phospholipase C activity.     

Glutamate Differently Modulates Metabotropic Glutamate Receptors in Neuronal and Glial Cells

Glutamate is an excitatory neurotransmitter implicated in learning and memory processes, but at high concentrations it acts as an excitotoxin causing degeneration and neuronal death. The aim of this work was to determine the excitotoxic effect of glutamate and the regulation of metabotropic glutamate receptors (mGluR) during excitotoxicity in neurons and C6 glioma cells. Results show that glutamate causes excitotoxic damage only in cortical neurons. Loss of cell viability in neurons was glutamate concentration- and time-dependent. Total mGluR levels were significantly reduced in these cells when exposed to glutamate. However, in C6 cells, which have been used as a model of glial cells, these receptors were regulated in a biphasic manner, decreased after 6 h, and increased after 24/48 h of treatment. Results show a cell dependent mGluR regulation by glutamate exposure which could mediate the vulnerability or not to glutamate mediated excitotoxicity.     

Phosphorylation and recruitment of Syk by immunoreceptor tyrosine-based activation motif-based phosphorylation of tamalin

Tamalin is a scaffold protein that forms a multiple protein assembly including metabotropic glutamate receptors (mGluRs) and several postsynaptic and protein-trafficking scaffold proteins in distinct mode of protein-protein association. In the present investigation, we report that tamalin possesses a typical immunoreceptor tyrosine-based activation motif (ITAM), which enables Syk kinase to be recruited and phosphorylated by the Src family kinases. Coimmunoprecipitation analysis of rat brain membrane fractions showed that tamalin is present in a multimolecular protein assembly comprising not only mGluR1 but also c-Src, Fyn, and a protein phosphatase, SHP-2. The protein association of both tamalin and c-Src, as determined by truncation analysis of mGluR1 in COS-7 cells, occurred at the carboxyl-terminal tail of mGluR1. Mutation analysis of tyrosine with phenylalanine in COS-7 cells revealed that paired tyrosines at the ITAM sequence of tamalin are phosphorylated preferentially by c-Src and Fyn, and this phosphorylation can recruit Syk kinase and enables it to be phosphorylated by the Src family kinases. The phosphorylated tyrosines at the ITAM sequence of tamalin were highly susceptible to dephosphorylation by protein-tyrosine phosphatases in COS-7 cells. Importantly, tamalin was endogenously phosphorylated and associated with Syk in retinoic acid-treated P19 embryonal carcinoma cells that undergo neuron-like differentiation. The present investigation demonstrates that tamalin is a novel signaling molecule that possesses a PDZ domain and a PDZ binding motif and mediates Syk signaling in an ITAM-based fashion.     

G protein-coupled receptor kinase-mediated desensitization of metabotropic glutamate receptor 1A protects against cell death

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.     

Type 2 metabotropic glutamate receptor (mGluR2) fails to negatively couple to cGMP in stably transfected cells

The group II metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3) share sequence homology, common pharmacology and negative coupling to cAMP. We recently discovered that mGluR3 also is negatively coupled through a G-protein to the cGMP transduction pathway in rat cerebellar granule cells and astrocytes. To test the hypothesis that mGluR2 also has access to the cGMP pathway, C6 glioma cells were stably transfected with mGluR2 and mGluR3 cDNA and their coupling to cGMP levels was characterized. In contrast to many other cell lines, C6 has a robust cGMP response that makes it attractive in the study of receptor coupling to this second messenger pathway. Consistent with prior studies, the mGluR3 receptor was negatively coupled to cGMP and this coupling was blocked by PTX. In contrast, mGluR2 agonists failed to reduce sodium nitroprusside stimulated cGMP levels in transfected cell lines where the receptor was negatively coupled to cAMP. These data provide further support for the functional divergence between these two closely related receptors.     

Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.     

Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway.

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with detergent-resistant membrane domains. Finally, clathrin-independent endocytosis requires dynamin and is specifically regulated by Rho family GTPases. These results define novel properties of receptor-mediated endocytosis and establish that the IL2 receptor is efficiently internalized through this clathrin-independent pathway.     

Co-localization and functional interaction between adenosine A(2A) and metabotropic group 5 receptors in glutamatergic nerve terminals of the rat striatum

The anti-Parkinsonian effect of glutamate metabotropic group 5 (mGluR5) and adenosine A(2A) receptor antagonists is believed to result from their ability to postsynaptically control the responsiveness of the indirect pathway that is hyperfunctioning in Parkinson's disease. mGluR5 and A(2A) antagonists are also neuroprotective in brain injury models involving glutamate excitotoxicity. Thus, we hypothesized that the anti-Parkinsonian and neuroprotective effects of A(2A) and mGluR5 receptors might be related to their control of striatal glutamate release that actually triggers the indirect pathway. The A(2A) agonist, CGS21680 (1-30 nM) facilitated glutamate release from striatal nerve terminals up to 57%, an effect prevented by the A(2A) antagonist, SCH58261 (50 nM). The mGluR5 agonist, CHPG (300-600 mum) also facilitated glutamate release up to 29%, an effect prevented by the mGluR5 antagonist, MPEP (10 microm). Both mGluR5 and A(2A) receptors were located in the active zone and 57 +/- 6% of striatal glutamatergic nerve terminals possessed both A(2A) and mGluR5 receptors, suggesting a presynaptic functional interaction. Indeed, submaximal concentrations of CGS21680 (1 nM) and CHPG (100 microm) synergistically facilitated glutamate release and the facilitation of glutamate release by 10 nM CGS21680 was prevented by 10 microm MPEP, whereas facilitation by 300 microm CHPG was prevented by 10 nM SCH58261. These results provide the first direct evidence that A(2A) and mGluR5 receptors are co-located in more than half of the striatal glutamatergic terminals where they facilitate glutamate release in a synergistic manner. This emphasizes the role of the modulation of glutamate release as a likely mechanism of action of these receptors both in striatal neuroprotection and in Parkinson's disease.     

Metabotropic glutamate receptor 5 modulates the nitric oxide-cGMP pathway in cerebellum in vivo through activation of AMPA receptors

Metabotropic glutamate receptors (mGluRs) modulate important processes in cerebellum including long-term depression, which also requires formation of nitric oxide (NO) and cGMP. Some reports suggest that mGluRs could modulate the NO-cGMP pathway in cerebellum. However this modulation has not been studied in detail. The aim of this work was to assess by microdialysis in freely moving rats whether activation of mGluR5 modulates the NO-cGMP pathway in cerebellum in vivo and to analyze the underlying mechanisms. We show that mGluR5 activation increases extracellular glutamate, citrulline and cGMP in cerebellum. Blocking NMDA receptors with MK-801 does not prevent any of these effects, indicating that NMDA receptors activation is not required. However in the presence of MK-801 the effects are more transient, returning faster to basal levels. Blocking AMPA receptors prevents the increase in citrulline and cGMP induced by mGluR5 activation, but not the increase in glutamate. The release of glutamate is prevented by tetrodotoxin but not by fluoroacetate, indicating that glutamate is released from neurons and not from astrocytes. Activation of AMPA receptors increases citrulline and cGMP. These data indicate that activation of mGluR5 induces an increase of extracellular glutamate which activates AMPA receptors, leading to activation of nitric oxide synthase and increased NO, which activates guanylate cyclase, increasing cGMP. The response mediated by AMPA receptors desensitize rapidly. Activation of AMPA receptors also induces a mild depolarization, allowing activation of NMDA receptors which prolongs the duration of the effect initiated by activation of AMPA receptors. These data support that the three types of glutamate receptors: mGluR5, AMPA and NMDA cooperate in the modulation of the grade and duration of activation of the NO-cGMP pathway in cerebellum in vivo. This pathway would modulate cerebellar processes such as long-term depression.     

Cellular Mechanisms of Protection by Metabotropic Glutamate Receptors During Anoxia and Nitric Oxide Toxicity

Abstract: Metabotropic glutamate receptors, nitric oxide (NO), and the signal transduction pathways of protein kinase C (PKC) and protein kinase A (PKA) can independently alter ischemic-induced neuronal cell death. We therefore examined whether the protective effects of metabotropic glutamate receptors during anoxia and NO toxicity were mediated through the cellular pathways of PKC or PKA in primary hippocampal neurons. Pretreatment with the metabotropic glutamate receptor agonists (±)-1-aminocyclopentane- trans -1,3-dicarboxylic acid, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD), and l (+)-2-amino-4-phosphonobutyric acid ( l -AP4) 1 h before anoxia or NO exposure increased hippocampal neuronal cell survival from ∼30 to 70%. In addition, posttreatment with 1 S ,3 R -ACPD or l -AP4 up to 6 h following an insult attenuated anoxic- or NO-induced neurodegeneration. In contrast, treatment with l -(+)-2-amino-3-phosphonopropionic acid, an antagonist of the metabotropic glutamate receptor, did not significantly alter neuronal survival during anoxia or NO exposure. Protection by the ACPD-sensitive metabotropic receptors, such as the subtypes mGluR1α, mGluR2, and mGluR5, appears to be dependent on the modulation of PKC activity. In contrast, l -AP4-sensitive metabotropic glutamate receptors, such as the subtype mGluR4, may increase neuronal survival through PKA rather than PKC. Thus, activation of specific metabotropic glutamate receptors is protective during anoxia and NO toxicity, but the signal transduction pathways mediating protection differ among the metabotropic glutamate receptor subtypes.     

Regulation of N-methyl-D-aspartic acid (NMDA) receptors by metabotropic glutamate receptor 7

Emerging evidence suggests that metabotropic glutamate receptors (mGluRs) are potential novel targets for brain disorders associated with the dysfunction of prefrontal cortex (PFC), a region critical for cognitive and emotional processes. Because N-methyl-D-aspartic acid receptor (NMDAR) dysregulation has been strongly associated with the pathophysiology of mental illnesses, we examined the possibility that mGluRs might be involved in modulating PFC functions by targeting postsynaptic NMDARs. We found that application of prototypical group III mGluR agonists significantly reduced NMDAR-mediated synaptic and ionic currents in PFC pyramidal neurons, which was mediated by mGluR7 localized at postsynaptic neurons and involved the β-arrestin/ERK signaling pathway. The mGluR7 modulation of NMDAR currents was prevented by agents perturbing actin dynamics and by the inhibitor of cofilin, a major actin-depolymerizing factor. Consistently, biochemical and immunocytochemical results demonstrated that mGluR7 activation increased cofilin activity and F-actin depolymerization via an ERK-dependent mechanism. Furthermore, mGluR7 reduced the association of NMDARs with the scaffolding protein PSD-95 and the surface level of NMDARs in an actin-dependent manner. These data suggest that mGluR7, by affecting the cofilin/actin signaling, regulates NMDAR trafficking and function. Because ablation of mGluR7 leads to a variety of behavioral symptoms related to PFC dysfunction, such as impaired working memory and reduced anxiety and depression, our results provide a potential mechanism for understanding the role of mGluR7 in mental health and disorders.     

Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors.

G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors. Whereas mGluR1 and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in many G protein-coupled receptors, is highly conserved among mGluRs, and may therefore not be involved in the specific recognition of G proteins in this receptor family. By constructing chimeric receptors between the AC-coupled mGluR3 and the PLC-coupled mGluR1c, we report here that both the C-terminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of PLC by mGluR1c. These two segments are rich in basic residues and are likely to be amphipathic alpha-helices, two characteristics of the G protein interacting domains of all G protein-coupled receptors. This indicates that whereas no amino acid sequence homology between mGluRs and the other G protein-coupled receptors can be found, their G protein interacting domains have similar structural features.