Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

Postsynaptic signaling at glutamatergic synapses as therapeutic targets

Dysregulation of glutamatergic synapses plays an important role in the pathogenesis of neurological diseases. In addition to mediating excitatory synaptic transmission, postsynaptic glutamate receptors interact with various membrane and intracellular proteins. They form structural and/or signaling synaptic protein complexes and thereby play diverse postsynaptic functions. Recently, several postsynaptic protein complexes have been associated with various neurological diseases and hence, have been characterized as important therapeutic targets. Moreover, novel small molecules and therapeutic peptides targeting and modulating the activities of these protein complexes have been discovered, some of which have advanced through preclinical translational research and/or clinical studies. This article describes the recent investigation of eight key protein complexes associated with the postsynaptic ionotropic and metabotropic glutamate receptors as therapeutic targets for central nervous system diseases.     

Molecular mechanisms underlying specificity of excitotoxic signaling in neurons

The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy and trauma has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Although calcium ions are considered key regulators of excitotoxicity, new evidence suggests that specific second messenger pathways rather than total Ca(2+) load, are responsible for mediating neuronal degeneration. Glutamate receptors are found localized at the synapse within electron dense structures known as the postsynaptic density (PSD). Localization at the PSD is mediated by binding of glutamate receptors to submembrane proteins such as actin and PDZ containing proteins. PDZ domains are conserved motifs that mediate protein-protein interactions and self-association. In addition to glutamate receptors PDZ-containing proteins bind a multitude of intracellular signal molecules including nitric oxide synthase. In this way PDZ proteins provide a mechanism for clustering glutamate receptors at the synapse together with their corresponding signal transduction proteins. PSD organization may thus facilitate the individual neurotoxic signal mechanisms downstream of receptors during glutamate overactivity. Evidence exists showing that inhibiting signals downstream of glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic insult. Furthermore we have shown that uncoupling the interaction between specific glutamate receptors from their PDZ proteins protects neurons against glutamate-mediated excitotoxicity. These findings have significant implications for the treatment of neurodegenerative diseases using therapeutics that specifically target intracellular protein-protein interactions.     

The trick of the tail: protein-protein interactions of metabotropic glutamate receptors

It was initially believed that G-protein-coupled receptors, such as metabotropic glutamate receptors, could simply be described as individual proteins that are associated with intracellular signal cascades via G-proteins. This view is no longer tenable. Today we know that metabotropic glutamate receptors (mGluRs) can dimerize and bind to a variety of proteins in addition to trimeric G-proteins. These newly identified protein interactions led to the discovery of new regulatory mechanisms that are independent of and sometimes synergistic with the classical G-protein-coupled second messenger pathways. Notably, several of these mechanisms connect mGluR-mediated signaling to other receptor classes, thereby creating a network of different receptor types and associated signal cascades. The intracellular C-termini of mGluRs play a key role in the regulation of these networks, and various new protein interactions of these domains were described recently. Because mGluRs are involved in a variety of physiological and pathophysiological processes, some of the proteins interacting with this receptor class have potential as valuable pharmaceutical targets. This review will give a comprehensive overview of proteins interacting with mGluR C-termini, highlight new evolving regulatory mechanisms for glutamatergic signal transduction and discuss possibilities for future drug development.     

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.     

Modulation of synaptic signalling complexes by Homer proteins

The number of neurotransmitter receptors in the postsynaptic membrane and their functional coupling to intracellular signalling cascades are important determinants of synaptic strength--and hence potential targets for plasticity related modulation. In this context, Homer/Vesl proteins have gained particular interest for three main reasons: (i) they constitute part of the molecular scaffold at postsynaptic densities of excitatory synapses in the mammalian brain; (ii) they physically link type-I metabotropic glutamate receptors to the postsynaptic density and to inositol 1,4,5-triphosphate receptors in the subsynaptic endoplasmic reticulum; and (iii) Homer-1a, which has been categorized as an immediate early gene isoform, exerts dominant-negative activity, suggesting that it is involved in activity dependent rearrangements at synaptic junctions. Although these fundamental aspects have been reviewed previously by Xiao et al., this review will address primarily more recent studies on the regulation of Homer 1a expression and on the role of Homer/Vesl proteins in spine morphogenesis and receptor targeting and signalling.     

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.     

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/X11beta/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.     

Activation of glutamate receptors inhibits Na/K-ATPase of cerebellum granule cells

Na/K-ATPase prepared from cerebellum granule cells of 10-12-day-old mice is inhibited by glutamate and its agonists, NMDA (ligand for ionotropic receptors) and ACPD (ligand for metabotropic receptors). The inhibition is specific and prevented by subsequent antagonists (MK-801 for ionotropic NMDA-receptors and MCPG for metabotropic receptors). The inhibiting effect of NMDA is significantly reversed by cysteine and that of ACPD by chelerythrine or indolyl maleimide. It is concluded that ionotropic receptors inhibit Na/K-ATPase because of intracellular production of reactive oxygen species, and metabotropic receptors mediate their effect via protein kinase C.     

Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA

Three major subtypes of glutamate receptors that are coupled to cation channels are known. Recently an additional subtype that is coupled to G proteins and stimulates inositol phospholipid metabolism (the metabotropic glutamate receptor) has been proposed. The pharmacological characteristics of this receptor have now been examined. Although it shares some agonists with N-methyl-D-aspartate- and quisqualate-subtype receptors, it shares virtually no antagonists with any of the three cation channel-coupled receptor subtypes. Thus the metabotropic glutamate receptor belongs to a receptor category that is completely different from that of the other three receptor subtypes, not only functionally, but also pharmacologically.     

Protein kinase Cbeta and delta selectively phosphorylate odorant and metabotropic glutamate receptors.

Recombinant protein segments from a metabotropic glutamate receptor and from an odorant receptor were used as substrates in protein kinase C phosphorylation assays. Protein kinase Cbeta and delta phosphorylated an intracellular consensus phosphorylation site in the metabotropic glutamate receptor. Only protein kinase Cdelta phosphorylated a novel extracellular consensus phosphorylation site in the odorant receptor. These results suggest differential regulation of these receptors by protein kinase C isotypes.     

Interaction of Vesl-1L/Homer 1c with syntaxin 13

Vesl-1S/Homer 1a, reported originally as Vesl/Homer, was isolated as a synaptic plasticity-regulated gene. The expression of Vesl-1S/Homer 1a is regulated during long-term potentiation in the hippocampus. Vesl-1L/Homer 1c, which appears to be formed by a splicing event, shares the N-terminus with Vesl-1S/Homer 1a and also contains additional amino acids at the C-terminus. The short form and the long form of the family members both interact with group 1 metabotropic glutamate receptors (mGluRs). We herein report the identification of syntaxin 13 as a molecule that interacts with Vesl-1L using yeast two-hybrid screening. Syntaxin 13 is a member of the syntaxin family and is regarded as soluble N-ethylmaleimide-sensitive attachment proteins receptors (SNAREs) in the endosomal membranes. The interaction of Vesl-1L and syntaxin 13 was biochemically confirmed by in vitro binding assays. The coexpression of the two proteins in the transfected cells resulted in a colocalization in the intracellular vesicle-like structures. We thus propose that the association of Vesl-1L with syntaxin 13 plays a role in the translocation of Vesl-1L to the intracellular organelles.     

N-Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells

Abstract: In previous studies, we demonstrated that the neuropeptide, N -acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1–6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans -1-aminocyclopentane-1,3-dicarboxylate ( trans -ACPD) and l -2-amino-4-phosphonobutyrate ( l -AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans -ACPD, activated each of the transfected receptors, whereas l -AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans -ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.     

How do G-protein-coupled receptors work? The case of metabotropic glutamate and GABA receptors

G-protein coupled receptors (GPCRs) represent the largest membrane proteins family in animal genomes. Being the receptors for most hormones and neurotransmitters, these proteins play a central role in intercellular communication. GPCRs can be classified into several groups based on the sequence similarity of their common structural feature: the heptahelical domain. The metabotropic receptors for the main neurotransmitters glutamate and gamma-aminobutyric acid (GABA) belong to the class III of GPCRs, together with others receptors for Ca2+, for sweet and amino acid taste compounds and for some pheromones, as well as for odorants in fish. Besides their transmembrane heptahelical domain responsible for G-protein activation, most of class III receptors possess a large extracellular domain responsible for ligand recognition. The recent resolution of the structure of this binding domain of one of these receptors, the mGlu1 receptor, together with the recent demonstration that these receptors are dimers, revealed an original mechanism of activation for these GPCRs. Such data open new possibilities to develop drugs aimed at modulating these receptors, and raised a number of interesting questions on the activation mechanism of other GPCRs.     

Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor-mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.