In silico binding affinity prediction for metabotropic glutamate receptors using both endpoint free energy methods and a machine learning-based scoring function.

Metabotropic glutamate receptors (mGluRs) play an important role in regulating glutamate signal pathways, which are involved in neuropathy and periphery homeostasis. mGluR4, which belongs to Group III mGluRs, is most widely distributed in the periphery among all the mGluRs. It has been proved that the regulation of this receptor is involved in diabetes, colorectal carcinoma and many other diseases. However, the application of structure-based drug design to identify small molecules to regulate the mGluR4 receptor is limited due to the absence of a resolved mGluR4 protein structure. In this work, we first built a homology model of mGluR4 based on a crystal structure of mGluR8, and then conducted hierarchical virtual screening (HVS) to identify possible active ligands for mGluR4. The HVS protocol consists of three hierarchical filters including Glide docking, molecular dynamic (MD) simulation and binding free energy calculation. We successfully prioritized active ligands of mGluR4 from a set of screening compounds using HVS. The predicted active ligands based on binding affinities can almost cover all the experiment-determined active ligands, with only one ligand missed. The correlation between the measured and predicted binding affinities is significantly improved for the MM-PB/GBSA-WSAS methods compared to the Glide docking method. More importantly, we have identified hotspots for ligand binding, and we found that SER157 and GLY158 tend to contribute to the selectivity of mGluR4 ligands, while ALA154 and ALA155 could account for the ligand selectivity to mGluR8. We also recognized other 5 key residues that are critical for ligand potency. The difference of the binding profiles between mGluR4 and mGluR8 can guide us to develop more potent and selective modulators. Moreover, we evaluated the performance of IPSF, a novel type of scoring function trained by a machine learning algorithm on residue-ligand interaction profiles, in guiding drug lead optimization. The cross-validation root-mean-square errors (RMSEs) are much smaller than those by the endpoint methods, and the correlation coefficients are comparable to the best endpoint methods for both mGluRs. Thus, machine learning-based IPSF can be applied to guide lead optimization, albeit the total number of actives/inactives are not big, a typical scenario in drug discovery projects.

Regulation and functional consequences of mGlu4 RNA editing.

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7 Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.

Activation mechanism of the G protein-coupled sweet receptor heterodimer with sweeteners and allosteric agonists.

The sweet taste in humans is mediated by the TAS1R2/TAS1R3 G protein-coupled receptor (GPCR), which belongs to the class C family that also includes the metabotropic glutamate and γ-aminobutyric acid receptors. We report here the predicted 3D structure of the full-length TAS1R2/TAS1R3 heterodimer, including the Venus Flytrap Domains (VFDs) [in the closed-open (co) active conformation], the cysteine-rich domains (CRDs), and the transmembrane domains (TMDs) at the TM56/TM56 interface. We observe that binding of agonists to VFD2 of TAS1R2 leads to major conformational changes to form a TM6/TM6 interface between TMDs of TAS1R2 and TAS1R3, which is consistent with the activation process observed biophysically on the metabotropic glutamate receptor 2 homodimer. We find that the initial effect of the agonist is to pull the bottom part of VFD3/TAS1R3 toward the bottom part of VFD2/TAS1R2 by ∼6 Šand that these changes get transmitted from VFD2 of TAS1R2 (where agonists bind) through the VFD3 and the CRD3 to the TMD3 of TAS1R3 (which couples to the G protein). These structural transformations provide a detailed atomistic mechanism for the activation process in GPCR, providing insights and structural details that can now be validated through mutation experiments.

Conformational dynamics of a class C G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in eukaryotes. Crystal structures have provided insight into GPCR interactions with ligands and G proteins, but our understanding of the conformational dynamics of activation is incomplete. Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders. A 'clamshell' ligand-binding domain (LBD), which contains the ligand-binding site, is coupled to the transmembrane domain via a cysteine-rich domain, and LBD closure seems to be the first step in activation. Crystal structures of isolated mGluR LBD dimers led to the suggestion that activation also involves a reorientation of the dimer interface from a 'relaxed' to an 'active' state, but the relationship between ligand binding, LBD closure and dimer interface rearrangement in activation remains unclear. Here we use single-molecule fluorescence resonance energy transfer to probe the activation mechanism of full-length mammalian group II mGluRs. We show that the LBDs interconvert between three conformations: resting, activated and a short-lived intermediate state. Orthosteric agonists induce transitions between these conformational states, with efficacy determined by occupancy of the active conformation. Unlike mGluR2, mGluR3 displays basal dynamics, which are Ca(2+)-dependent and lead to basal protein activation. Our results support a general mechanism for the activation of mGluRs in which agonist binding induces closure of the LBDs, followed by dimer interface reorientation. Our experimental strategy should be widely applicable to study conformational dynamics in GPCRs and other membrane proteins.

Purification of Stabilized GPCRs for Structural and Biophysical Analyses.

G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor toward a specific physiologically relevant conformational state. The resultant stabilized receptor (known as a StaR) can be purified in multiple-milligram quantities, whilst retaining correct folding, thus enabling the generation of reagents suitable for a broad range of structural and biophysical studies. Example protocols for the purification of StaR proteins for analysis, ligand screening with the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), surface plasmon resonance (SPR), and crystallization for structural studies are presented.

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families.

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1-3% of the world's population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.

Allosteric Binding Site and Activation Mechanism of Class C G-Protein Coupled Receptors: Metabotropic Glutamate Receptor Family.

Metabotropic glutamate receptors (mGluR) are mainly expressed in the central nervous system (CNS) and contain eight receptor subtypes, named mGluR1 to mGluR8. The crystal structures of mGluR1 and mGluR5 that are bound with the negative allosteric modulator (NAM) were reported recently. These structures provide a basic model for all class C of G-protein coupled receptors (GPCRs) and may aid in the design of new allosteric modulators for the treatment of CNS disorders. However, these structures are only combined with NAMs in the previous reports. The conformations that are bound with positive allosteric modulator (PAM) or agonist of mGluR1/5 remain unknown. Moreover, the structural information of the other six mGluRs and the comparisons of the mGluRs family have not been explored in terms of their binding pockets, the binding modes of different compounds, and important binding residues. With these crystal structures as the starting point, we built 3D structural models for six mGluRs by using homology modeling and molecular dynamics (MD) simulations. We systematically compared their allosteric binding sites/pockets, the important residues, and the selective residues by using a series of comparable dockings with both the NAM and the PAM. Our results show that several residues played important roles for the receptors' selectivity. The observations of detailed interactions between compounds and their correspondent receptors are congruent with the specificity and potency of derivatives or compounds bioassayed in vitro. We then carried out 100 ns MD simulations of mGluR5 (residue 26-832, formed by Venus Flytrap domain, a so-called cysteine-rich domain, and 7 trans-membrane domains) bound with antagonist/NAM and with agonist/PAM. Our results show that both the NAM and the PAM seemed stable in class C GPCRs during the MD. However, the movements of "ionic lock," of trans-membrane domains, and of some activation-related residues in 7 trans-membrane domains of mGluR5 were congruent with the findings in class A GPCRs. Finally, we selected nine representative bound structures to perform 30 ns MD simulations for validating the stabilities of interactions, respectively. All these bound structures kept stable during the MD simulations, indicating that the binding poses in this present work are reasonable. We provided new insight into better understanding of the structural and functional roles of the mGluRs family and facilitated the future structure-based design of novel ligands of mGluRs family with therapeutic potential.

Computational analysis of the extracellular domain of the Ca²âº-sensing receptor: an alternate model for the Ca²âº sensing region.

The extracellular Ca(2+) sensing receptor (CaSR) belongs to Class C G-protein-coupled receptors (GPCRs) which include receptors for amino acids, γ-aminobutyric acid and glutamate neurotransmitters. CaSR has been described as having an extended sequence containing a Ca(2+) binding pocket within an extracellular amino (N)-terminal domain, called a Venus Fly Trap (VFT) module. CaSR is thought to consist of three domains: 1) a Ca(2+-)sensory domain, 2) a region containing 7 transmembrane (TM) helices, and 3) a carboxy (C)-terminal tail. We find that SPOCTOPUS (a combination of hidden Markov models and artificial neural networks) predicts that Homo sapiens CaSR contains two additional TM helices ((190)D - G(210); (262)S-E(282)), with the second TM helix containing a pore-lining region ((265)K - I(280)). This predicts that the putative Ca(2+) sensory domain is within an extracellular loop, N-terminal to the highly conserved heptahelical bundle. This loop contains both the cysteine-rich domain ((537)V - C(598)) and a 14 residue "linker" sequence ((599)I - F(612)) thought to support signal transmission to the heptahelical bundle. Thus domain 1 may contain a 189 residue N-terminal extracellular region followed successively by TM-1, a short intracellular loop, TM-2 and a 329 residue extracellular loop; rather than the proposed 620 residue VFT module based on crystallography of the N-terminal region of mGluR1. Since the topologies of the two proteins differ, the published CaSR VFT model is questionable. CaSR also contains multiple caveolin-binding motifs and cholesterol-binding (CRAC/CARC) domains, facilitating localization to plasma membrane lipid rafts. Ion sensing may involve combination of pore-lining regions from CaSR dimers and CaSR-bound caveolins to form ion channels capable of monitoring ionized Ca(2+) levels.

Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer.

G protein-coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.

Location-dependent signaling of the group 1 metabotropic glutamate receptor mGlu5.

Although G protein-coupled receptors are primarily known for converting extracellular signals into intracellular responses, some receptors, such as the group 1 metabotropic glutamate receptor, mGlu5, are also localized on intracellular membranes where they can mediate both overlapping and unique signaling effects. Thus, besides "ligand bias," whereby a receptor's signaling modality can shift from G protein dependence to independence, canonical mGlu5 receptor signaling can also be influenced by "location bias" (i.e., the particular membrane and/or cell type from which it signals). Because mGlu5 receptors play important roles in both normal development and in disorders such as Fragile X syndrome, autism, epilepsy, addiction, anxiety, schizophrenia, pain, dyskinesias, and melanoma, a large number of drugs are being developed to allosterically target this receptor. Therefore, it is critical to understand how such drugs might be affecting mGlu5 receptor function on different membranes and in different brain regions. Further elucidation of the site(s) of action of these drugs may determine which signal pathways mediate therapeutic efficacy.

Somatic mutations in GRM1 in cancer alter metabotropic glutamate receptor 1 intracellular localization and signaling.

The activity of metabotropic glutamate receptors (mGluRs) is known to be altered as the consequence of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease. However, little attention has been paid to this receptor family's potential link with cancer. Recent reports indicate altered mGluR signaling in various tumor types, and several somatic mutations in mGluR1a in lung cancer were recently described. Group 1 mGluRs (mGluR1a and mGluR5) are coupled primarily to Gαq, leading to the activation of phospholipase C and to the formation of diacylglycerol and inositol 1,4,5-trisphosphate, leading to the release of Ca(2+) from intracellular stores and protein kinase C (PKC) activation. In the present study, we investigated the intracellular localization and G protein-dependent and -independent signaling of eight GRM1 (mGluR1a) somatic mutations. Two mutants found in close proximity to the glutamate binding domain and cysteine-rich region (R375G and G396V) show both decreased cell surface expression and basal inositol phosphate (IP) formation. However, R375G shows increased ERK1/2 activation in response to quisqualate stimulation. A mutant located directly in the glutamate binding site (A168V) shows increased quisqualate-induced IP formation and, similar to R375G, increased ERK1/2 activation. Additionally, a mutation in the G protein-coupled receptor kinase 2/PKC regulatory region (R696W) shows decreased ERK1/2 activation, whereas a mutation within the Homer binding region in the carboxyl-terminal tail (P1148L) does not alter the intracellular localization of the receptor, but it induces changes in cellular morphology and exhibits reduced ERK1/2 activation. Taken together, these results suggest that mGluR1a signaling in cancer is disrupted by somatic mutations with multiple downstream consequences.

Optimization of an ether series of mGlu5 positive allosteric modulators: molecular determinants of MPEP-site interaction crossover.

We report the optimization of a series of non-MPEP site metabotropic glutamate receptor 5 (mGlu(5)) positive allosteric modulators (PAMs) based on a simple acyclic ether series. Modifications led to a gain of MPEP site interaction through incorporation of a chiral amide in conjunction with a nicotinamide core. A highly potent PAM, 8v (VU0404251), was shown to be efficacious in a rodent model of psychosis. These studies suggest that potent PAMs within topologically similar chemotypes can be developed to preferentially interact or not interact with the MPEP allosteric binding site.

Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome.

Enhanced metabotropic glutamate receptor subunit 5 (mGluR5) function is causally associated with the pathophysiology of fragile X syndrome, a leading inherited cause of intellectual disability and autism. Here we provide evidence that altered mGluR5-Homer scaffolds contribute to mGluR5 dysfunction and phenotypes in the fragile X syndrome mouse model, Fmr1 knockout (Fmr1(-/y)). In Fmr1(-/y) mice, mGluR5 was less associated with long Homer isoforms but more associated with the short Homer1a. Genetic deletion of Homer1a restored mGluR5-long Homer scaffolds and corrected several phenotypes in Fmr1(-/y) mice, including altered mGluR5 signaling, neocortical circuit dysfunction and behavior. Acute, peptide-mediated disruption of mGluR5-Homer scaffolds in wild-type mice mimicked many Fmr1(-/y) phenotypes. In contrast, Homer1a deletion did not rescue altered mGluR-dependent long-term synaptic depression or translational control of target mRNAs of fragile X mental retardation protein, the gene product of Fmr1. Our findings reveal new functions for mGluR5-Homer interactions in the brain and delineate distinct mechanisms of mGluR5 dysfunction in a mouse model of cognitive dysfunction and autism.

Interdomain movements in metabotropic glutamate receptor activation.

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.

Estrogen receptors stimulate brain region specific metabotropic glutamate receptors to rapidly initiate signal transduction pathways.

Estradiol and other steroid hormones modulate the nervous system and behavior on both acute and long-term time scales. Though estradiol was originally characterized as a regulator of gene expression through the action of nuclear estrogen receptors (ERs) that directly bind DNA, research over the past thirty years has firmly established that estradiol can bind to extra-nuclear ERs associated with the cellular membrane, producing changes in neurons through stimulation of various intracellular signaling pathways. Several studies have determined that the classical ERs, ERα and ERß, mediate some of these fast-acting signaling pathways through activation of G proteins. Since ERα and ERß are not G protein-coupled receptors, the mechanisms by which ERs can stimulate signal transduction pathways are a focus of recent research. Here we discuss recent studies illustrating one mechanism by which ERα and ERß initiate these pathways: through direct association with metabotropic glutamate receptors (mGluRs). Estradiol binding to these membrane-localized estrogen receptors results in mGluR signaling independent of glutamate. ERs are organized with mGluRs into functional signaling microdomains via caveolin proteins. The pairing of ERs to specific mGluRs via caveolins is region specific, with ERs being linked to different mGluRs in hippocampal, striatal, and other neurons. It is becoming clear that ER signaling through mGluRs is one important mechanism by which estrogens can modulate neuron and glial physiology, ultimately impacting various aspects of nervous system function.

Metabotropic glutamate receptors: from the workbench to the bedside.

Metabotropic glutamate (mGlu) receptors were discovered in the mid 1980s and originally described as glutamate receptors coupled to polyphosphoinositide hydrolysis. Almost 6500 articles have been published since then, and subtype-selective mGlu receptor ligands are now under clinical development for the treatment of a variety of disorders such as Fragile-X syndrome, schizophrenia, Parkinson's disease and L-DOPA-induced dyskinesias, generalized anxiety disorder, chronic pain, and gastroesophageal reflux disorder. Prof. Erminio Costa was linked to the early times of the mGlu receptor history, when a few research groups challenged the general belief that glutamate could only activate ionotropic receptors and all metabolic responses to glutamate were secondary to calcium entry. This review moves from those nostalgic times to the most recent advances in the physiology and pharmacology of mGlu receptors, and highlights the role of individual mGlu receptor subtypes in the pathophysiology of human disorders. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

An integrated view on the role of receptor mosaics at perisynaptic level: focus on adenosine A(2A), dopamine D(2), cannabinoid CB(1), and metabotropic glutamate mGlu(5) receptors.

The available evidence for receptor-receptor interactions between adenosine A(2A), dopamine D(2), cannabinoid CB(1), and metabotropic glutamate mGlu(5) receptors (A(2A), D(2), CB(1), and mGlu(5), respectively) is revised under the "receptor mosaic" perspective. Furthermore, the concept of "hub receptor" is defined in accordance with informatics and it is tentatively illustrated in the case of the hypothesized tetramer formed by the above mentioned receptors. On the basis of some biochemical features of the four receptors and of a bioinformatics analysis, an objective deduction of their "similarity" has been obtained. To this aim the Canberra, Euclidean and Chebyshev multivariate distance metrics have been used. It is interesting to note that A(2A) and D(2) are the most different ones, while CB(1) and mGlu(5) are the most similar ones among the four receptors analyzed. Finally, by means of a bioinformatics analysis based on different approaches the possible binding sites mediating G-protein-coupled receptor (GPCR) interactions have been indicated. It is interesting to note that in some instances accordance has been found between the bioinformatics indications and the available experimental data.

Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization.

Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state. The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission. Here, we report experiments designed to investigate mGluR kinetics in real-time. We inserted either CFP or YFP into the second intracellular loop of mGluR1beta. When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC(50) of 7.5 microM. The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators. These results suggest that the FRET response reports active conformations of mGluR1 dimers. The solution exchange at the cell membrane was optimized for voltage-clamped cells by recording the current induced by co-application of 30 mM potassium. When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (approximately 50 ms). During long-lasting applications, no desensitization was observed. In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms. Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC(50) and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission.

Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher-order oligomers in living cells.

G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu(5) receptors (mGlu(5)R) and dopamine D(2) receptors (D(2)R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu(5)R, D(2)R and adenosine A(2A) receptor (A(2A)R). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu(5)R, D(2)R and A(2A)R in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu(5)R/D(2)R/A(2A)R oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.

Regions of the amino terminus of the P2X receptor required for modification by phorbol ester and mGluR1alpha receptors.

The potentiation of P2X(1) receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1alpha receptors was sensitive to inhibition of novel forms of protein kinase C. Potentiation was also reduced by co-expression of an amino terminal P2X(1) receptor minigene. Cysteine point mutants of residues Tyr(16)-Gly(30) were expressed in Xenopus oocytes. Peak current amplitudes to ATP for Y16C, T18C and R20C mutants were reduced, however this did not result from a decrease in surface expression of the channels. The majority of the mutants showed changes in the time-course of desensitization of ATP evoked currents indicating the important role of this region in regulation of channel properties. PMA and mGluR1alpha potentiation was abolished for the mutants Y16C, T18C, R20C, K27C and G30C. Minigenes incorporating either Y16C, K27C, V29C or G30C still inhibited PMA responses. However D17C, T18C or R20C mutant minigenes were no longer effective suggesting that these residues are important for interaction with regulatory factors. These results demonstrate that the conserved YXTXK/R sequence and a region with a conserved glycine residue close to the first transmembrane segment contribute to PMA and GPCR regulation of P2X(1) receptors.