Ligand--protein interactions in the glutamate receptor

Fourier transform infrared spectroscopy was used to investigate ligand-protein interactions in the ligand-binding domain of the GluR4 glutamate receptor subunit. Glutamate binding induces more extensive secondary structural changes in the ligand-binding domain than does kainate binding. Glutamate also alters the hydrogen bonding strength of the single free cysteine side chain in the domain, while kainate does not. On the other hand, the interaction of a binding site arginine residue with kainate appears to be stronger than that with glutamate. These results identify chemical and structural differences that may explain the different functional characteristics of the two agonists acting on ionotropic glutamate receptors. In doing so, they complement and extend recent crystallographic structures of the ligand-binding domain.     

Synthesis and pharmacological characterisation of 2,4-dicarboxy-pyrroles as selective non-competitive mGluR1 antagonists

Metabotropic glutamate receptors (mGluRs) are an unusual family of G-protein coupled receptor (GPCR), and are characterised by a large extracellular N-terminal domain that contains the glutamate binding site. We have identified a new class of non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonists, 2,4-dicarboxy-pyrroles which are endowed with nanomolar potency. They interact within the 7 transmembrane (7TM) domain of the receptor and show antinociceptive properties when tested in a number of different animal models.     

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.     

Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

Recently three orphan G-protein coupled receptors, RAIG1, GPRC5B and GPRC5C, with homology to members of family C (metabotropic glutamate receptor-like) have been identified. Using the protein sequences of these receptors as queries we identified overlapping expressed sequence tags which were predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane. The four human receptor subtypes, which we assign to group 5 of family C GPCRs, show 31-42% amino acid sequence identity to each other and 20-25% sequence identity to the transmembrane domains of metabotropic glutamate receptor subtypes 2 and 3 and other family C members. In contrast to the remaining family C members, the group 5 receptors have short amino terminal domains of some 30-50 amino acids. GPRC5D was shown to be clustered with RAIG1 on chromosome 12p13.3 and like RAIG1 and GPRC5B to consist of three exons, the first exon being the largest containing all seven transmembrane segments. GPRC5D mRNA is widely expressed in the peripheral system but all four receptors show distinct expression patterns. Interestingly, mRNA levels of all four group 5 receptors were found in medium to high levels in the kidney, pancreas and prostate and in low to medium levels in the colon and the small intestine, whereas other organs only express a subset of the genes. In an attempt to delineate the signal transduction pathway(s) of the orphan receptors, a series of chimeric receptors containing the amino terminal domain of the calcium sensing receptor or metabotropic glutamate receptor subtype 1, and the seven transmembrane domain of the orphan receptors were constructed and tested in binding and functional assays.     

Amino acid recognition by Venus flytrap domains is encoded in an 8-residue motif

A motif foramino acid recognition by proteins or domains of the periplasmic binding protein-like I superfamily has been identified. An initial pattern of 5 residues was based on a multiple sequence alignment of selected proteins of that fold family and on common structural features observed in the crystal structure of some members of the family [leucine isoleucine valine binding protein (LIVBP), leucine binding protein (LBP), and metabotropic glutamate receptor type 1 (mGlu1R) amino terminal domain)]. This pattern was used against the PIR-NREF sequence database and further refined to retrieve all sequences of proteins that belong to the family and eliminate those that do not belong to it. A motif of 8 residues was finally selected to build up the general signature. A total of 232 sequences were retrieved. They were found to belong to only three families of proteins: bacterial periplasmic binding proteins (PBP, 71 sequences), family 3 (or C) of G-protein coupled receptor (GPCR) (146 sequences), and plant putative ionotropic glutamate receptors (iGluR, 15 sequences). PBPs are known to adopt a bilobate structure also named Venus flytrap domain, or LIVBP domain in the present case. Family 3/C GPCRs are also known to hold such a domain. However, for plant iGluRs, it was previously detected by classical similarity searches but not specifically described. Thus plant iGluRs carry two Venus flytrap domains, one that binds glutamate and an additional one that would be a modulatory LIVBP domain. In some cases, the modulator binding to that domain would be an amino acid.     

Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha

The extracellular domain of the metabotropic glutamate receptor 1alpha (mGluR1alpha) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1alpha's interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1alpha labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.     

Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.     

A novel site on the Galpha -protein that recognizes heptahelical receptors

Specific domains of the G-protein alpha subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between alpha4 and alpha5 helices of the G-protein alpha subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Galpha(15) and its human homologue Galpha(16), whereas metabotropic glutamate receptor 8 activated Galpha(15) only. The extreme C-terminal 20 amino acid residues are identical between the Galpha(15) and Galpha(16) and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Galpha(16) that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of Galpha(16). One module comprises alpha4 helix, alpha4-beta6 loop (L9 Loop), beta6 sheet, and alpha5 helix. The other, not described previously, is located within the loop that links the N-terminal alpha helix to the beta1 strand of the Ras-like domain of the alpha subunit. Coupling of Galpha(16) protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully.     

Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.     

Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.     

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.     

Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors

The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.     

Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.     

Glutamate Acts as a Partial Inverse Agonist to Metabotropic Glutamate Receptor with a Single Amino Acid Mutation in the Transmembrane Domain

Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain is severely limited, in contrast to the well studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 transmembrane domain with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the hydrogen-bonding network by T789Y^6.43 mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y^6.43 suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Tyr-789^6.43 and Gly-831^7.45 in T789Y^6.43 mutant was important for this phenotype. Therefore, the present study is consistent with the notion that the metabotropic glutamate receptor shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.     

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.     

Modulation of glutamate release from parallel fibers by mGlu4 and pre-synaptic GABA(A) receptors

The regulation of pre-synaptic glutamate release is important in the maintenance and fidelity of excitatory transmission in the nervous system. In this study, we report a novel interaction between a ligand-gated ion channel and a G-protein coupled receptor which regulates glutamate release from parallel fiber axon terminals. Immunocytochemical analysis revealed that GABA(A) receptors and the high affinity group III metabotropic glutamate receptor subtype 4 (mGlu4) are co-localized on glutamatergic parallel fiber axon terminals in the cerebellum. GABA(A) and mGlu4 receptors were also found to co-immunoprecipitate from cerebellar membranes. Independently, these two receptors have opposing roles on glutamate release: pre-synaptic GABA(A) receptors promote, while mGlu4 receptors inhibit, glutamate release. However, coincident activation of GABA(A) receptors with muscimol and mGlu4 with the agonist (2S)-S-2-amino-4-phosphonobutanoic acid , increased glutamate release from [(3) H]glutamate-loaded cerebellar synaptosomes above that observed with muscimol alone. Further support for an interaction between GABA(A) and mGlu4 receptors was obtained in the mGlu4 knockout mouse which displayed reduced binding of the GABA(A) ligand [(35) S]tert-butylbicyclophosphorothionate, and decreased expression of the α1, α6, β2 GABA(A) receptor subunits in the cerebellum. Taken together, our data suggest a new role for mGlu4 whereby simultaneous activation with GABA(A) receptors acts to amplify glutamate release at parallel fiber-Purkinje cell synapses.     

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.     

Structure of the metabotropic glutamate receptor

In the twelve years since the molecular elucidation of the metabotropic glutamate receptor subtype 1, a class III family of G-protein-coupled receptors has emerged; members of this family include the calcium-sensing receptor, the GABA(B) receptor, some odorant receptors and some taste receptors. Atomic structures of the ligand-binding core of the original metabotropic glutamate receptor 1 obtained using X-ray crystallography provide a foundation for determining the initial receptor activation of this important family of G-protein-coupled receptors.