Calcium Waves Through Group I Metabotropic Glutamate Receptors

1. Classification of mGluRs

Metabotropic glutamate receptors (mGluRs) belong to Family C G-protein-coupled receptor superfamily. Typically, a mGluR has a large extracellular domain of over 500 amino acid residues comprising a signalling peptide, venus flytrap domain (VFD) which captures bound agonist, and cysteine-rich domain (CRD) which conjoins VFD and TMs. In humans and rodents, eight subtypes of mGluRs have been identified. These are divided into three groups based on similarities in primary structures as below:

Group I    mGluR1 and mGluR5;
Group II    mGluR2 and mGluR3; and
Group III    mGluR4, mGluR6, mGluR7 and mGluR8.

Group I receptors predominantly couple to Gq/11 which increases intracellular calcium concentration ([Ca2+]i) by releasing the divalent cation stored in cell organelles. A range of protein kinase C (PKC) is also activated in contributing oscillatory changes of [Ca2+]i. It has been demonstrated for mGluR5 that the receptor propagates calcium waves through dynamic desensitisation involving negative feedback by PKC; the oscillation frequency was determined by receptor density rather than agonist concentration (Nash et al. 2002).

As for mGluR1a, the signalling is sensitive to agonist concentration; the expression level of Gq/11 could also determine threshold for the onset of the calcium signal generation (Atkinson et al. 2006). PKC isozymes which respond to both DAG and elevated [Ca2+]i were shown to repetitively translocate between cytosol and plasma membrane in response to mGluR1a activation; notably, PKCα, PKCβI and PKCβII exhibited different patterns with the former two translocating at basal levels, whereas PKCβII responded only to the agonist-induced mGluR1a activation (Babwah et al. 2003).

Group II and Group III members have tendencies coupling to Gi/0 which down-regulate adenylyl cyclase activity.

In neurones, Group I mGluRs tend to localise at postsynaptic density, Groups II members can be found either pre- or post-synaptic, whereas Group III members have slight tendencies to express at the presynaptic side.

All mGluRs have an inherent tendency to exist as dimers and oligomerisation is a prerequisite in receptor activation process (reviewed by Pin et al 2009).


2. Ligands

2.1. Endogenous agonist: L-glutamate

L-glutamate is an excitatory neurotransmitter that modulate conductivity of three types of ionotropic receptors: N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and kainate receptor. Glu binding to the ionotropic receptors immediately alters the cellular ionic state. On the other hand, mGluR activation modulates cellular machinery of protein synthesis and degradation, fine tuning coordinated intracellular calcium signalling of intricate networks; overall producing more dynamic, prolonged effects on the cells.

2.2. Synthetic ligands

Listed below are some of synthetic ligands often used to study group I mGluRs. Each compound is identifiable at sources: ‘Chemspider ID’ for ChemSpider; “Compound ID” for PubChem; and <Compound ID> for ChEMBL, given at the end of each description.

Agonists:

(S)-3,5-dihydroxyphenylglycine (DHPG) is an orthosteric agonist with selectivity to group I mGluRs displaying equivalent potencies at mGluR1 and mGluR5 ‘11377133’.

2-chloro-5-hydroxyphenylglycine (CHPG) is a weak agonist selective to mGluR5 ‘10654595’.

Positive Allosteric Modulators:

Butyl(9H-xanthene-9-carbonyl)carbamate (Ro67-4853) potentiates mGluR1 “CID9949202”.

4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (VU-29) potentiates mGluR5 <CHEMBL411221>.

3,3'-difluorobenzaldazine (DFB) potentiates mGluR5 “CID 6604893”. It is a full agonist of the mGluR5 without its extracellular domain (Goudet et al., 2003).

N-(4-Chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl)-2-hydroxybenzamide (CPPHA) potentiates mGluR5 “CID9931205”.

Antagonists:

4-[(2S)-2-amino-1-hydroxy-1-oxopropan-2-yl]benzoic acid (LY367385) is relatively selective to mGluR1 <CHEMBL257626>.

Negative Allosteric Modulators:

(-)-ethyl(7E)-7-hydroxyimino-1,7a-dihydrocyclopropa[b]chromene-1a-carboxylate (CPCCOEt) is a mGluR1-selective negative allosteric modulator <CHEMBL337583>.

3-[(2-Methyl-4-thiazolyl)ethynyl]pyridine (MTEP) acts at mGluR5 ‘7969985’.

2-Methyl-6-(phenylethynyl)pyridine (MPEP) competes with VU-29 at mGluR5 ‘2291589’. NB: It acts as inverse agonist in the absence of the extracellular domain (Goudet et al., 2004).


3. Intracellular Signalling via Group I mGluRs

The Gq/11 and intracellular calcium release
Glutamate binding to mGluR1 or -5 triggers signal pathways initiated by Gq/11: phospholipase Cβ hydrolyses phosphoinositides to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); IP3 increases [Ca2+]i by releasing the cellular reservoir, whilst DAG activates PKC isozymes (α, βI, βII, γ, δ, ε, η, θ) met; the activity of some PKC isozymes (α, βI, βII, γ) are also sensitively induced by changes in [Ca2+]i. 

3a. Adaptors/interacting proteins

NHERF2
At its C-tail, mGluR5 can interact with PDZ domain of NHERF2 for more effective Gq-signalling with prolonged calcium mobilisation; the two were found to colocalise notably in astrocytic processes and postsynaptic neurones (Paquet et al 2006). NHERF2 prevents mGluR5-coupling to calcium channels (Cav2.2) which generate N-type currents, but it has no effect on potassium channels (Kv7) for M-type currents (Filipov et al 2010).   

Calmodulin
A mutagenesis study on rodent Group I mGluRs showed that calmodulin (CaM) interacts favourably with the C-terminal Leu residue (L896 in rat mGluR5a) of mGluR5. A weaker interaction between CaM and the corresponding Val residue (V909 in rat mGluR1) of mGluR1 was enhanced by the Leu-substitution of the residue; in the reciprocal mGluR5 mutant, the disruption of CaM-mGluR5 association decreased the cell-surface expression of the receptor in hippocampal neurones (Choi, Chung & Roche 2011).

Homers and Shanks
Certain isoforms of group I mGluRs interact with Homer family of adaptor proteins, via a Pro-Pro-x-x-Phe motif (where x is unspecified) in the C-terminal domain (Tu JC et al. 1998; Beneken et al. 2000). In mammals, three Homer proteins, Homer1, Homer2 and Homer3, can exist in isoforms of various lengths; some longer forms are previously known postsynaptic density (PSD) proteins, Cupidin (a.k.a. Homer 2a, 2b) and PSD-Zip45 (a.k.a. Homer 1c); Homer proteins can link the mGluRs with specific calcium channels, actin cytoskeletons, Dynamin 3, and other scaffolding proteins such as Shanks (reviewed by Shiraishi-Yamaguchi & Furuichi 2007). Amongst PSD Shank proteins, SSTRIP (somatostatin receptor-interacting protein) specifically interacts with the C-tail region of SSTR2 (Zitzer et al. 1999).

Some Kinases
mGluR5 has been shown to co-immunoprecipitate with epidermal growth factor receptor (EGFR) following DHPG-simulation in rodent astrocytes (Peavy et al. 2001). Such interactions may have been made possible by the adaptor proteins; by interacting with Shank bound to receptor tyrosine kinase (RTK), Homers may link mGluR signals with ERK-MAPK and PI3K pathways. Shank3 was shown to associate with a longer isoform of RET, Ret9 (Schuetz et al. 2004). Although there is no evidence for Shank binding to EGFR, a genomic analysis showed an increase in copy numbers of EGFR and Shank2 in squamous cell carcinoma (Carneiro et al., 2008), possibly implying their association. In a speculative thought one might assume that receptor tyrosine kinases and mGluR5 might be brought together via Homer/Shank crossing over the different receptors.


4. The local and immediate regulation of Group I mGluR activity

Both mGluR1 and mGluR5 are subjected to phosphorylation by PKCs: mGluR1a at intracellular loop (ICL) 2, likely by PKCδ or PKCβ (Medler & Bruch 1999; Francesconi & Duvoisin 2000) and mGluR5 at ICL1, ICL2, or its C-tail for internalisation (Gereau & Heinemann 1998).

Phosphorylation of activated mGluR5 at S901 by PKC altered propagation patterns of calcium oscillatory waves, deterred CaM binding to the receptor, and reduced cell-surface expression of the receptor (Lee et al. 2008).

PKCε was shown to negatively regulate mGluR5-induced calcium oscillation in astrocytes; inhibiting protein phosphatase 1/2A produced a similar effect (Bradley & Challiss 2011).

Group I mGluRs can be subjected to be degradation via ubiquitination. Seven in absentia homolog 1A (Siah1A) can induce this (Moriyoshi et al. 2004).


5. Group I mGluRs with ionotropic receptors

Activation of potassium channels
ERK signalling through mGluR5 upregulates A-type currents generated by potassium channels with Kv4.2 subunit (Hu et al. 2007). The effect was observed in excitatory dorsal horn neurones, wherein colocalisation of Kv4.2 and PKCγ was coincided (Hu & Gereau 2011).

NMDAR on mGluR5 pharmacology
Orthosteric agonist (CHPG) and positive allosteric modulators (DFB, CDPPB) of mGluR5 could augment hippocampal field potentials induced by NMDA through PKC activation; but when pretreated with NMDA, the effect was only produced by the orthosteric agonist (Chen, Liao and Chan 2011).

The effect of mGluR5 on NMDAR-medicated synaptic transmission
MPEP, a mGluR5-selective antagonist, reduced long-term synaptic depression (LTD) of NMDAR-induced excitatory post-synaptic current (EPSC) (Harney et al. 2006). 

The Effect of mGluR Activation on AMPAR Internalisation
AMPA endocytosis could be enhanced by mGluR5-CaM interaction (Choi, Chung & Roche 2011). It has also been reported that dephosphorylation at Tyr of an AMPAR subunit, GluA2, caused endocytosis (Gladding et al 2009a); this can be done by striatal-enriched tyrosine phosphatase (STEP) (Zhang et al. 2008) of which expression is upregulated by mGluRs through MAPK and PI3K pathways (discussed in review by Gladding et al 2009b).  


6. Synaptic plasticity modulated by mGluRs

Long-term synaptic potentiation (LTP)
In addition to rapid calcium influxes though NMDAR, mGluRs also appear to contribute to the induction of LTP. In CA3-CA1 hippocampal regions, astrocytes release glutamate which increases excitatory postsynaptic current potentiation via NMDAR; the glutamate spontaneously released by astrocytes thereby facilitates LTP. EPSC could be reduced by gliotoxin fluorocitrate, calcium chelator, or group I mGluR antagonists; the blocking the mGluRs resulted in less frequent miniature synaptic response (Bonansco et al. 2011).

Long-term synaptic depression (LTD)
In cortical and hippocampal neurones, mGluRs are involved in induction of LTD (Otani & Connor 1998; Cho et al 2000). In CA1 region, mGluR5 agonist DHPG induced LTD; it was inhibited by antagonist MPEP (1 μM). The induction of LTD involved p38 MAPK (Moult et al. 2008). LTD, which is induced by DHPG or PP-LFS and depends on protein-synthesis in juvenile CA1, requires ERK rather than p38 MAPK (Gallagher et al. 2004).

Elevating [Ca2+]i transients could result in either long-term synaptic potentiation (LTP) or LTD. The latter requires both mGluR action and retrograde influence reflecting altered postsynaptic [Ca2+]i levels: activation of voltage-gated Ca2+ channels prior to mGluR activation induced PLC-dependent synthesis of endocannabinoids which propagate retrograde signal to favour LTD (Nevian & Sakmann 2006).

CB1R in mGluR-induced LTD
Robust LTD was induced by selective mGluR5 agonist CHPG, with CB1R activation appeared to occur downstream (Izumi & Zorumski 2011). In response to glutamate stimulation, endocannabimoids (eCB) are released as retrograde messengers which target CB1R to support LTD (Robbe et al. 2002; Sjöström et al. 2003). In the nucleus accumbens, cocaine blocks the eCB-retrograde signalling; the effect accompanied enhanced Homer expression and reduction in mGluR5 cell-surface expression (Fourgeaud et al. 2004).

CB1R and Gq in LTD
LTDs could arise in various forms; autaptic (i.e. self-sensing) LTD was shown to require CB1R, as well as iGluRs and group I mGluRs, in cultured hippocampul neurones. CB1R exhibits this effect, neither via Gi/0 nor Gs, but via PLC and Ca2+ release, suggesting involvement of Gq (Kellogg et al. 2009). Atypical Gq-coupling of CB1 in response to a synthetic agonist WIN55,212-2 has previously been documented (Lauckner et al. 2005).

CB1R antagonists prevent mGluR-induced epleptiform and LTD
DHPG causes prolonged epileptiform in the CA3 region. Application of CB1 antagonists SR141716 or AM251 significantly prevented the DHPG-induced epileptiform, but the effect was little once it has been induced. Exposing the brain slices with DHPG and SR141716 prevented LTD and EPSP remained at or above control level (Karr et al. 2010).       


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