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Three clones of PC12 cells that differ with respect to their nerve growth factor (NGF) receptors were examined: wild-type PC12 cells that have both trkA and p75LNTR receptors; the MR-1 clone that possesses a normal trkA receptor and a truncated form of p75LNTR without the extracellular NGF-binding part; and a new PC12 variant, called v-clone, that is partly characterized here. The v-clone had no demonstrable binding to trkA, but displayed binding to p75LNTR as assessed by chemical crosslinking. NGF did not induce any change in the tyrosine phosphorylation of phosphatidy-3'-kinase in the v-clone. NGF induced neurite extension in wild-type cells, induced it more rapidly in mR-1, but not at all in v-clone cells. The v-clone lacked the b-form of protein kinase C, but transfection with this enzyme did not restore responsiveness to NGF. Neurite extension in response to staurosporine and basic fibroblast growth factor was equal in wild-type and v-clone cells. All three clones responded to forskolin, with the mR-1 clone the most responsive. NGF stimulated AP 1 binding activity in all clones. The response was transient in the MR-1 clone but prolonged in the wild-type and v-clone cells. In the wild-type and MR-1 clone cells, AP 1 binding activity was reduced by a tyrphostin analog, whereas in the v-clone cells it was inhibited by staurosporine. NGF increased inositol (1,4,5)-trisphosphate (InsP3) formation in all clones. In the wild-type and v-clone cells the InsP3 responses were followed by [Ca2+]i increases. It is concluded that although trkA is required for differentiation in response to NGF in PC12 cells, the concomitant stimulation, by NGF, of p75LNTR may affect phospholipase C and AP 1. This may be important for the reported ability of p75LNTR to modify the phenotypic changes induced in PC12 cells by NGF.

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na+ channels with tetrodotoxin. We found that 8-pCPT-2′-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the β-adrenergic receptor (βAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of βARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that βARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals. Background: G protein-coupled receptors generating cAMP at nerve terminals modulate neurotransmitter release. Results: β-Adrenergic receptor enhances glutamate release via Epac protein activation and Munc13-1 translocation at cerebrocortical nerve terminals. Conclusion: Protein kinase A-independent signaling pathways triggered by β-adrenergic receptors control presynaptic function. Significance: β-Adrenergic receptors target presynaptic release machinery.

There is serious interest in understanding the dynamics of the receptor–receptor and receptor–protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor–receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor–receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology.

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons.

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

Rabs comprise the largest member of the Ras superfamily of small GTPases with over 60 proteins in mammals and 11 proteins in yeast. Like all small GTPases, Rabs oscillate between an inactive GDP-bound conformation and an active GTP-bound state that is tethered to lipid membranes via a C-terminal prenylation site on conserved cysteine residues. In their active state, Rabs regulate various aspects of membrane trafficking, including vesicle formation, transport, docking, and fusion. The critical element of biological activity is the recruitment of cytosolic effector proteins to specific endomembranes by active Rabs. The importance of Rabs in cellular processes is apparent from their links to genetic disorders, immunodeficiency, cancer, and pathogen invasion. During the last decade, numerous structures of complexes have shed light on the molecular basis for Rab/effector specificity and their topological organization on subcellular membranes. Here, I review the known structures of Rab/effector complexes and their modes of oligomerization. This is followed by a brief discussion on the thermodynamics of effector recruitment, which has not been documented sufficiently in previous reviews. A summary of diseases associated with Rab/effector trafficking pathways concludes this chapter.

This chapter provides a theoretical framework on the quantitative description of protein aggregation. The reader is provided with an overview of the fundamental theory of linear and helical polymers, as well as an introduction on the parameters governing evolution of aggregates over time. The models presented for the interpretation of the protein aggregation process take into account the contributions of different physicochemical parameters such as charge, hydrophobicity, and secondary structure propensity. Finally, we discuss our current understanding of how prediction of aggregation rates and identification of aggregation-prone protein regions are predicted from the information contained in the primary amino acid sequence.

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

The Dnmt3a DNA cytosine-C5 methyltransferase has been recently shown to exhibit a complex oligomerization and multimerization potential, the structural basis and functional implications of which will be the subject of this contribution. The enzyme forms a linear heterotetramer with Dnmt3L, in which the interaction of Dnmt3a and 3L stimulates the catalytic activity of Dnmt3a. Isolated Dnmt3a forms protein filaments that bind to several DNA molecules oriented in parallel, which plays an essential role in the location of the enzyme to heterochromatin. Dnmt3L disrupts Dnmt3a protein filaments and leads to a redistribution of the enzyme in cells toward euchromatin. Finally, Dnmt3a complexes and Dnmt3a/3L heterotetramers cooperatively multimerize on DNA forming protein–DNA filaments. This leads to a preference of the enzyme for periodic methylation of DNA and supports its heterochromatic localization.

Amyloid fibrils are highly organized and generally insoluble protein aggregates rich in β secondary structure that can be formed by a wide range of sequences. They have been the object of intense scrutiny because their formation has been associated with a number of neurodegenerative disorders such as Alzheimer’s, Parkinson’s, Huntington’s, and Creutzfeldt–Jakob’s diseases. As a consequence of these efforts, much is now known about the properties of proteins that render them prone to form amyloid fibrils, about the mechanism of fibrillation, about the molecular structures of the fibrils, and about the forces that stabilize them. The relationship between the structural properties of the monomeric protein and those of the corresponding aggregate has been, in particular, intensively studied. In this chapter, we will provide an account of current knowledge on this intriguing relationship and provide the reader with key references about this topic.

Conformational Protein Diseases (CPDs) comprise over forty clinically and pathologically diverse disorders in which specific altered proteins accumulate in cells or tissues of the body. The most studied are Alzheimerβ's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses. They are characterised by three dimensional conformational alterations, which are often rich in β- structure. Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with like protein molecules. The misfolded proteins can impart their anomalous properties to soluble, monomeric proteins with the same amino acid sequence by a process that has been likened to seeded crystallization. However, these potentially pathogenic proteins also have important physiological actions, which have not completely characterized. This opens up the question of what process transforms physiological actions into pathological actions and most intriguing, is why potentially dangerous proteins have been maintained during evolution and are present from yeasts to humans. In the present paper, we introduce the concept of mis-exaptation and of mis-tinkering since they may help in clarifying some of the double edged sword aspects of these proteins. Against this background an original interpretative paradigm for CPDs will be given in the frame of the previously proposed Red Queen Theory of Aging.

The A1 adenosine receptor from pig brain cortex has been identified by means of two antipeptide antibodies against two domains of the receptor molecule: PC/10 antiserum was raised against a part of the third intracellular loop, and PC/20 antiserum was raised against a part of the second extracellular loop. PC/10 antibody was able to recognize a 39-kDa band that corresponded to the Al receptor, as demonstrated by immunoblotting and by immunoprecipitation of the molecule cross-linked to [125I](R)-2-azidoN2-p-hydroxy(phenylisopropyl)adenosine. Besides the 39-kDa band, PC/20 also recognized a 74-kDa form that does not seem to correspond to a receptor-G protein complex. The occurrence of the two bands was detected and analyzed in samples from different species and tissues showing a heterogeneous distribution of both. The 74-kDa form can be converted into the 39-kDa form by treatment with agonists or antagonists of Al adenosine receptors. These results suggest that A1 adenosine receptor can occur in dimers and that the dimer–monomer conversion might be regulated by adenosine as the physiological ligand. Since the 74-kDa aggregates were not recognized by PC/10, it is likely that part of the third intracellular loop participates in the protein–protein interaction. ©1995 Wiley-Liss, Inc.

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity.

Galanin receptor (GalR) subtypes1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein coupled receptors (GPCRs) in the Central Nervous System (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with transinhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression.

GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C-terminal tail, in particular on a cystein-enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine-rich domain is linked to the previously described receptor-mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C-terminal cysteine-rich domain protected against GPR37-mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C-terminal tail cysteine-rich domain, which played a critical role in receptor cell surface expression, function and GPR37-mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinson's disease) of this rather unknown member of the GPCR family. GPR37 is a rather unknown orphan GPCR that in a genetic form of Parkinson's disease accumulates intracellularly and induces neuronal death. In this study, we describe a cystein-rich domain in the C-terminal tail of the receptor that controls its membrane expression and whose absence reduces the GPR37-dependent citotoxic effects on ER stress and cell death.

In the CNS, an antagonistic interaction has been shown between adenosine A(2A) and dopamine D(2) receptors (A(2A)Rs and D(2)Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor-receptor interaction have recently been explored, as the fine tuning of this target (namely the A(2A)R/D(2)R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer-based approach to examine the allosteric modulation of the D(2)R within the A(2A)R/D(2)R oligomer and the dependence of this receptor-receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D(2)R. Interestingly, we observed a negative allosteric effect of the D(2)R agonist quinpirole on A(2A)R ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217-222 and 267-269) on intracellular loop 3 of the D(2)R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor-receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A(2A)R/D(2)R oligomer in living cells.

Polymorphic variants of the dopamine D 4 receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D 4.4) and the 2-repeat (D 4.2) variants form functional heteromers with the short isoform of the dopamine D 2 receptor (D 2S), the 7-repeat risk allele (D 4.7) does not. D 2 receptor activation in the D 2S-D 4 receptor heteromer potentiates D 4 receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D 4.7 or in the striatum of knockin mutant mice carrying the 7 repeats of the human D 4.7 in the third intracellular loop of the D 4 receptor. In the striatum, D 4 receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D 2S receptors. This interaction shows the same qualitative characteristics than the D 2S-D 4 receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D 2S receptor activation potentiates D 4 receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D 2S-D 4.7 heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD. Fil: González, S.. Universidad de Barcelona; España Fil: Rangel Barajas, C.. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; México Fil: Peper, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Lorenzo Lopez, Juan Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Moreno, E.. Universidad de Barcelona; España Fil: Ciruela, F.. Universidad de Barcelona; España Fil: Borycz, J.. National Institutes of Health; Estados Unidos Fil: Ortiz, J.. Universitat Autònoma de Barcelona; España Fil: Lluís, C.. Universidad de Barcelona; España Fil: Franco, R.. Universidad de Navarra; España Fil: McCormick, P. J.. Universidad de Barcelona; España Fil: Volkow, N. D.. National Institutes of Health; Estados Unidos Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Floran, B.. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; México Fil: Ferré, S.. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; México

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. Within situproximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1–5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1–5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons.

Understanding of the molecular mechanisms of protein–protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed to characterize plasma membrane PPIs. Importantly, these technologies alone – or in concert with complementary methods (i.e. SNAP-tag/TR-FRET, TIRF/FRET) – can become extremely powerful approaches for visualizing cell surface PPIs, even between more than two proteins and also in native tissues. Interestingly, these methods would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Accordingly, herein we provide a thorough assessment on all biotechnological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied in the study of PPIs occurring at the cell surface of living cells.

Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks.

Adenosine is a nucleoside distributed throughout the entire organism as an intermediary metabolite. At the extracellular level, adenosine plays multiple physiologic roles, interacting with specific receptors: A1, A2A, A2B and A3 (Fredholm et al., 2001). While the A1Rs and A3Rs are coupled in an inhibitory way to adenylate cyclase through the Gi/o protein, the A2Rs are coupled in a stimulatory way to this enzymatic activity through Gs protein (Ralevic & Burnstock, 1998). Adenosine levels are increased after ischemia, hypoxia, excitotoxicity, inflammation and cerebral lesions. In these situations, it is considered that high adenosine levels play a neuroprotective role (Ribeiro et al., 2002). Interestingly, adenosine regulates the release of glutamate, the main excitatory neurotransmitter of the nervous system (Sebastiao & Ribeiro, 1996). A1Rs are widely expressed in the brain and have been shown to modulate neuronal excitability by decreasing pre-synaptic release of various neurotransmitters (Fredholm & Dunwiddie, 1988). The most dramatic inhibitory actions are on the glutamatergic system (Masino et al., 2002). In the central nervous system (CNS), A1Rs are associated with neuroprotective processes (Angulo et al., 2003; Dunwiddie and Masino, 2001). Moreover, they are upregulated in human neurodegenerative diseases with abnormal protein aggregates and it is related to compensatory mechanisms (Albasanz et al., 2007, 2008; Angulo et al., 2003; Perez-Buira et al., 2007; Rodríguez et al., 2006). Regarding A2ARs, these receptors are concentrated in the striatum, modulating dopaminergic activity, but they are also present in the hippocampus and cerebral cortex, modulating the glutamate release in the brain. Adenosine activity through A2 receptors (A2ARs) can eventually give rise to neurotoxicity, neuronal damage and cellular death (de Mendoça et al., 2000). In fact, A2ARs activity is associated with the outcome of cerebral injury as well as the development of Abeta- induced synaptotoxicity (Canas et al., 2009; Cunha, 2005; Stone et al., 2009).

It is proposed that the moonlighting concept can be applied to G protein coupled receptors (GPCRs) as, obviously, they can carry out different types of functions. The same motifs in, for example, the third intracellular loop, can moonlight by switching between receptor-receptor interactions and interactions with signaling proteins such as G proteins or calmodulin. A "guide-and-clasp" manner of receptor-receptor interactions has been proposed where the "adhesive guides" may be the triplet homologies. As an example, the triplets AAR (or RAA) and AAE (or EAA) homologies in A(2A) R-D2 R heteromers may guide-and-clasp binding not only of the two protomers but also of calmodulin and G(i) . A beautiful moonlighting phenomenon in the A(2A) R-D2 R heteromer is that the positively charged D2 R N-terminal third intracellular loop epitope (VLRRRRKRVN) may switch between bindings to the negatively charged A(2A) R epitope (SAQEpSQGNT), localized in the medium segment of the C terminus of the A2A receptor to several negative epitopes of calmodulin. Furthermore, overlapping motifs may favor moonlighting to G(i/o) via inter alia electrostatic interaction between triplets AAR(in D2 R third intracellular loop) and AAE (G(i/alpha1) ) (and/or their symmetric variants) contributing to guide-and-clasp D2 R-G(i) interactions Thus, moonlighting in GPCR heteromers can take place via allosteric receptor-receptor interactions and is also described in D1 R-D2 R, D2 R-5-HT2 R,and A1 R-P2Y1 heteromers. Allosteric receptor-receptor interactions in GPCR-receptor tyrosine kinases (RTKs) heteromers and postulated ion channel receptor-RTK heteromers-like, for example, AMPA-NMDA-TrkB heteromers may lead to moonlighting of the participating GPCR and RTK protomers altering, for example, the pattern of the five major signaling pathways of the RTKs favoring MAPK and/or mTOR signaling with high relevance for neurodegenerative processes and depression induced atrophy of neurons. Moonlighting may also develop in the intracellular loops and C-terminal of the GPCRs as a result of dynamic allosteric interactions between different types of G proteins and other receptor interacting proteins in these domains of the receptor.

γ-Aminobutyric acid type B (GABAB) receptors play a critical role in neuronal excitability and modulation of synaptic neurotransmission in the central nervous system. They are G protein-coupled receptors that signal primarily through activation of G proteins (i.e. pertussis toxin sensitive Gαi/o family) to modulate the function of inwardly-rectifying K⁺ and voltage-gated Ca²⁺ channels, and to trigger cyclic adenosine monophosphate cascades. Functional GABAB receptors are obligated heterodimers formed by the co-assembly of two subunits, the GABAB1 and the GABAB2, which interact via coiled-coil domains in their C-terminal tails. It is now quite well established that GABAB receptors interact not only with heterotrimeric G proteins and effector ion channels but also with a plethora of accessory proteins that might impinge into the receptor's biology. Indeed, these proteins have been implicated in several key functional aspects of the receptor, namely to link functional GABAB receptors with components of the relevant signalling pathways, to target the receptor into specific subcellular compartments, to participate in their trafficking to and from the plasma membrane, and to regulate their signalling properties. Overall, in this review we focus on those proteins that interact with GABAB receptors. Thus, understanding how the interaction between GABAB receptors and its accessory proteins takes place will definitively open new opportunities for pharmacological tool assessment of novel therapeutic targets for the treatment of several neurological diseases involving these receptors.

Voltage sensitivity has been demonstrated for some GPCRs. At the dopamine D(2S) receptor, this voltage sensitivity is agonist-specific; some agonists, including dopamine, exhibit decreased potency at depolarized potentials, whereas others are not significantly affected. In the present study, we examined some of the receptor-agonist interactions contributing to these differences, and investigated how dopamine D(2S) receptor voltage sensitivity affects clinically used dopamine agonists. GIRK channel activation in voltage-clamped Xenopus oocytes was used as readout of receptor activation. Structurally distinct agonists and complementary site-directed mutagenesis of the receptor's binding site were used to investigate the role of agonist-receptor interactions. We also confirmed that the depolarization-induced decrease of dopamine potency in GIRK activation is correlated by decreased binding of radiolabeled dopamine, and by decreased potency in G protein activation. In the mutagenesis experiments, a conserved serine residue as well as the conserved aspartate in the receptor's binding site were found to be important for voltage sensitive potency of dopamine. Furthermore, the voltage sensitivity of the receptor had distinct effects on different therapeutic D(2) agonists. Depolarization decreased the potency of several compounds, whereas for others, efficacy was reduced. For some agonists, both potency and efficacy were diminished, whereas for others still, neither parameter was significantly altered. The present work identifies some of the ligand-receptor interactions which determine agonist-specific effects of voltage at the dopamine D(2S) receptor. The observed differences between therapeutic agonists might be clinically relevant, and make them potential tools for investigating the roles of dopamine D(2) receptor voltage sensitivity in native tissue.

Adenosine receptors are G protein-coupled receptors (GPCRs) that mediate the physiological functions of adenosine. In the central nervous system adenosine A(2A) receptors (A(2A)Rs) are highly enriched in striatopallidal neurons where they form functional oligomeric complexes with other GPCRs such us the dopamine D(2) receptor (D(2)R). Furthermore, it is assumed that the formation of balanced A(2A)R/D(2)R receptor oligomers are essential for correct striatal function as the allosteric receptor-receptor interactions established within the oligomer are needed for properly sensing adenosine and dopamine. Interestingly, A(2A)R activation reduces the affinity of striatal D(2)R for dopamine and the blockade of A(2A)R with specific antagonists facilitates function of the D(2)R. Thus, it may be postulated that A(2A)R antagonists are pro-dopaminergic agents. Therefore, A(2A)R antagonists will potentially reduce the effects associated with dopamine depletion in Parkinson's disease (PD). Accordingly, this class of compounds have recently attracted considerable attention as potential therapeutic agents for PD pharmacotherapy as they have shown potential effectiveness in counteracting motor dysfunctions and also displayed neuroprotective and anti-inflammatory effects in animal models of PD. Overall, we provide here an update of the current state of the art of these A(2A)R-based approaches that are under clinical study as agents devoted to alleviate PD symptoms.

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.2)R, D(4.4)R and D(4.7)R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D(4)R biogenesis and quality control in the endoplasmic reticulum plays an important role for D(4)R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D(4.4)R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D(4)R. The different polymorphic repeat variants of the D(4)R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D(4)Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.

The concept of intra-membrane receptor–receptor interactions (RRIs) between different types of G protein-coupled receptors (GPCRs) and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 through the biochemical analysis of the effects of neuropeptides on the binding characteristics of monoamine receptors in membrane preparations from discrete brain regions and functional studies of the interactions between neuropeptides and monoamines in the control of specific functions such as motor control and arterial blood pressure control in animal models.Whether GPCRs can form high-order structures is still a topic of an intense debate. Increasing evidence, however, suggests that the hypothesis of the existence of high-order receptor oligomers is correct. A fundamental consequence of the view describing GPCRs as interacting structures, with the likely formation at the plasma membrane of receptor aggregates of multiple receptors (Receptor Mosaics) is that it is no longer possible to describe signal transduction simply as the result of the binding of the chemical signal to its receptor, but rather as the result of a filtering/integration of chemical signals by the Receptor Mosaics (RMs) and membrane-associated proteins. Thus, in parallel with experimental research, significant efforts were spent in bioinformatics and mathematical modelling. We review here the main approaches that have been used to assess the interaction interfaces allowing the assembly of GPCRs and to shed some light on the integrative functions emerging from the complex behaviour of these RMs. Particular attention was paid to the RMs generated by adenosine A2A, dopamine D2, cannabinoid CB1, and metabotropic glutamate mGlu5 receptors (A2A, D2, CB1 and mGlu5, respectively), and a possible approach to model the interplay between the D2–A2A–CB1 and D2–A2A–mGlu5 trimers is proposed. This article is part of a Special Issue entitled: “Adenosine Receptors”.

The existence of a supramolecular organization of the G protein-coupled receptor (GPCR) is now being widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlies this phenomenon still remains unsolved. Recently, it has been proposed that a trans-conformational switching model could be the mechanism allowing direct inhibition/activation of receptor activation/inhibition, respectively. Thus, heterotropic receptor-receptor allosteric regulations are behind the GPCR oligomeric function. In this paper we want to revise how GPCR oligomerization impinges on several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. In particular, the rationale of receptor oligomerization might lie in the need of sensing complex whole cell extracellular signals and translating them into a simple computational model.

A better understanding of the molecular mechanisms of receptor–receptor interactions is essential to comprehend the functional meaning of the phenomenon of G protein-coupled receptor (GPCR) oligomerization. In this chapter we discuss how new methodological strategies derived from non-invasive fluorescence- and luminescence-based approaches (i.e. FRET, BRET, BiFC/BiLC) have been successfully used in the study of GPCR oligomerization and consequently provided new insights into the occurrence of GPCR oligomers in living cells. These technologies alone, or in concert with complimentary methods (i.e. SRET, BRET/BiFC and SNAP-tag/TR-FRET), can be extremely powerful approaches for visualizing receptor–receptor interactions—even between more than two proteins and also in native tissues. Here we provide a thorough assessment on all biotechnological aspects of these fluorescence/luminescence-based methodologies, including the strengths and weaknesses, when applied in the study of GPCR oligomerization.

It has been estimated that at least 50% of the drugs available on the market act on G-protein coupled receptors (GPCRs) and most of these are basically or agonists or antagonists of this type of receptors. Herein, we propose new putative targets for drug development based on recent data on GPCR allosterism and on the existence of receptor mosaics (RMs). The main target for drug development is still GPCRs, but the focus is not the orthosteric binding pocket. According to the mosaic model of the plasma membrane, we mainly discuss the possibility of indirect modulatory pharmacological actions on expression/function of GPCRs. In particular, the following two new targets will be analyzed: a) The possibility of pharmacological interventions on the roamer-type of volume transmission (VT), which allow the intercellular transfer of set of signal molecules such as GPCRs, tetraspanins and ribonucleic acids. Thus, there is the possibility of pharmacological interventions on the decoding capabilities of neurons and/or glial cells by means of an action on composition and release of micro-vesicles. b) The possibility of pharmacological interventions on epigenetic mechanisms by taking into account their inter-relationships with GPCRs. As a matter of fact, there are epigenetic changes that are characteristic of periods of developmental plasticity that could provide a target for therapeutic intervention in the event of brain damage. We believe that almost all the biochemical knowledge presently available on GPCRs can be used in the development of these new pharmacological approaches.

While the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last fifteen years, the existence of a multi-receptor complex involving direct receptor–receptor interactions, called receptor oligomers, begins to be widely accepted. Eventually, it has been postulated that oligomers constitute a distinct functional form of the GPCRs with essential receptorial features. Also, it has been proven, under certain circumstances, that the GPCR oligomerization phenomenon is crucial for the receptor biosynthesis, maturation, trafficking, plasma membrane diffusion, and pharmacology and signalling. Adenosine receptors are GPCRs that mediate the physiological functions of adenosine and indeed these receptors do also oligomerize. Accordingly, adenosine receptor oligomers may improve the molecular mechanism by which extracellular adenosine signals are transferred to the G proteins in the process of receptor transduction. Importantly, these adenosine receptor-containing oligomers may allow not only the control of the adenosinergic function but also the fine-tuning modulation of other neurotransmitter systems (i.e. dopaminergic and glutamatergic transmission). Overall, we underscore here recent significant developments based on adenosine receptor oligomerization that are essential for acquiring a better understanding of neurotransmission in the central nervous system under normal and pathological conditions. This article is part of a Special Issue entitled: “Adenosine Receptors”.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or tar- geting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associ- ated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC. © The Author 2011. Published by Oxford University Press.

Recent experimental studies demonstrate that membrane composition is altered in certain human disorders. Modifying membrane composition (e.g. varying the concentration of cholesterol or certain phospholipid species) could in turn affect the behaviour of transmembrane proteins such as G protein-coupled receptors (GPCRs). To study the impact that an altered membrane structure has on GPCR aggregation, we performed long-timescale molecular dynamics simulations of GPCR oligomers, a complex thought to be involved in different diseases (e.g. central nervous system disorders). Interestingly, our simulations show that the aggregation of GPCRs is highly dependent on structural changes at the membrane level. To corroborate this effect, we have subsequently carried out bioluminescence resonance energy transfer (BRET) experiments in cells. Preliminary BRET results show that GPCR aggregation is significantly affected by the composition of cell membranes. Whereas the alteration of certain membrane properties (such as membrane thickness) have already been proposed to affect the activity of transmembrane proteins [1], here we report, for the first time, the key role of specific membrane components in the modulation of GPCR aggregation.Reference[1] Dacosta et. al A distinct mechanism for activating uncoupled nicotinic acetylcholine receptors. Nature chemical biology, September 2013.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Aims Atrial fibrillation (AF) is associated with abnormal sarcoplasmic reticulum (SR) calcium release, which is promoted by adenosine A2A receptor (A2AR) activation. Here, we tested the hypothesis that abnormal calcium release in AF is linked to A2AR remodelling. Methods and results Western blotting and quantitative real-time PCR were used to determine A2AR mRNA and protein levels in right atrial samples from patients with and without AF. Effects of A2AR activation on calcium handling were assessed with patch-clamp technique and confocal calcium imaging. A2AR mRNA levels and functional A2ARs were moderately up-regulated in patients with atrial dilation and markedly up-regulated in those with AF. Accordingly, A2AR stimulation significantly increased ryanodine receptor phosphorylation in AF patients, and spontaneous calcium waves increased moderately in myocytes from patients with atrial dilation and strongly in patients with AF (2.2 ± 2.1 to 14.3 ± 8.8 min−1, n = 6, P = 0.01). Moreover, the high baseline level of calcium waves in AF was reduced by A2AR antagonists (3.5 ± 2.0 to 1.3 ± 1.3 min−1, n = 6, P = 0.007) or adenosine deaminase (1.7 ± 1.5 to 0.5 ± 0.6 min−1, n = 10, P = 0.02) suggesting that A2ARs are activated by endogenous adenosine. Indeed, intracellular perfusion with adenosine significantly increased the calcium wave frequency (1.1 ± 0.8 to 8.2 ± 3.3 min−1, n = 8), whereas adenosine removal from the cytosol decreased it (2.1 ± 0.9 to 0.3 ± 0.3 min−1, n = 8, P = 0.04). Conclusions Atrial fibrillation patients show increased A2AR expression that may account for the high baseline level of spontaneous SR calcium release seen in myocytes from these patients, and the ability of A2AR antagonists to reduce this abnormal calcium release points to the A2AR as a novel molecular target in AF.

Given that coactivation of adenosine A(2A) (A(2A)R) and dopamine D(2) (D(2)R) receptors results in the coaggregation, cointernalization, and codesensitization of the A(2A)R and D(2)R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A(2A)R agonist CGS21680 in A(2A)R-D(2)R-coexpressing cells to modulate the D(2)R agonist-induced recruitment of β-arrestin2 to the D(2)R by means of proximity-based bioluminescence resonance energy transfer (BRET(2)) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET(2) signal between β-arrestin2(RLuc) and D(2L)R(GFP2) upon D(2)R activation, by increasing the potency of the D(2)R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2(GFP2) and D(2L)R(YFP) as seen from the co-trafficking analysis. Furthermore, the A(2A)R agonist advanced the time for the increase in Akt phosphorylation obtained with the D(2)R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A(2A)R-D(2)R heteromers. Taken together, the results indicate that the antagonistic A(2A)R-D(2)R allosteric receptor-receptor interaction in A(2A)R-D(2)R heteromers favors β-arrestin2 recruitment to the D(2L)R protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.

In view of the co-distribution of dopamine D(₂L)R and 5-hydroxytryptamine 5-HT(₂A) receptors (D(₂L)R and 5-HT(₂A)R, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D(₂L)R-5-HT(₂A)R heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D(₂L)R and the 5-HT(₂A)R form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D(₂L)R-5-HT(₂A)R heteromeric signaling was analyzed we found that the 5-HT(₂A)R-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D(₂L)R as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D(2L)R-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D(₂L)R and 5-HT(₂A)R within the heteromer led to inhibition of the D(₂L)R functioning, thus suggesting the existence of a 5-HT(₂A)R-mediated D(₂L)R trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D₂R-5-HT(₂A)R may be involved in the receptor interface. Overall, the presence of the D(₂L)R-5-HT(₂A)R heteromer in discrete brain regions is postulated based on the existence of D(₂L)R-5-HT(₂A) receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.

The stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABA(B) ) receptors, activates G protein-gated inwardly-rectifying K(+) (GIRK) channels, which influence membrane excitability. There is now evidence suggesting that G protein-coupled receptors and G protein-gated inwardly-rectifying K(+) [GIRK/family 3 of inwardly-rectifying K(+) (Kir3)] channels do not diffuse freely within the plasma membrane, but instead there are direct protein-protein interactions between them. Here, we used bioluminescence resonance energy transfer, co-immunoprecipitation, confocal and electron microscopy techniques to investigate the oligomerization of GABA(B) receptors with GIRK channels containing the GIRK3 subunit, whose contribution to functional channels is still unresolved. Co-expression of GABA(B) receptors and GIRK channels in human embryonic kidney-293 cells in combination with co-immunoprecipitation experiments established that the metabotropic receptor forms stable complexes with GIRK channels. Using bioluminescence resonance energy transfer, we have shown that, in living cells under physiological conditions, GABA(B) receptors interact directly with GIRK1/GIRK3 heterotetramers. In addition, we have provided evidence that the receptor-effector complexes are also found in vivo and identified that the cerebellar granule cells are one neuron population where the interaction probably takes place. Altogether, our data show that signalling complexes containing GABA(B) receptors and GIRK channels are formed shortly after biosynthesis, probably in the endoplasmic reticulum and/or endoplasmic reticulum/Golgi apparatus complex, suggesting that this might be a general feature of receptor-effector ion channel signal transduction and supporting a channel-forming role for the GIRK3 subunit.

Based on indications of direct physical interactions between neuropeptide and monoamine receptors in the early 1980s, the term receptor-receptor interactions was introduced and later on the term receptor heteromerization in the early 1990s. Allosteric mechanisms allow an integrative activity to emerge either intramolecularly in G protein-coupled receptor (GPCR) monomers or intermolecularly via receptor-receptor interactions in GPCR homodimers, heterodimers, and receptor mosaics. Stable heteromers of Class A receptors may be formed that involve strong high energy arginine-phosphate electrostatic interactions. These receptor-receptor interactions markedly increase the repertoire of GPCR recognition, signaling and trafficking in which the minimal signaling unit in the GPCR homomers appears to be one receptor and one G protein. GPCR homomers and GPCR assemblies are not isolated but also directly interact with other proteins to form horizontal molecular networks at the plasma membrane.

Evidence exists that the adenosine receptor A 2A R and the dopamine receptor D 2 R form constitutive heteromers in living cells. Mass spectrometry and pull-down data showed that an arginine-rich domain of the D 2 R third intracellular loop binds via electrostatic interactions to a specific motif of the A 2A R C-terminal tail. It has been indicated that the phosphorylated serine 374 might represent an important residue in this motif. In the present study, it was found that a point mutation of serine 374 to alanine reduced the A 2A R ability to interact with D 2 R. Also, this point mutation abolished the A 2A R-mediated inhibition of both the D 2 R high affinity agonist binding and signaling. These results point to a key role of serine 374 in the A 2A R–D 2 R interface. All together these results indicate that by targeting A 2A R serine 374 it will be possible to allosterically modulate A 2A R–D 2 R function, thus representing a new approach for therapeutically modulate D 2 R function.

Opioids provide effective analgesia in adult patients with painful inflammatory diseases. The proposed mechanism of action is the activation of peripheral opioid receptors, which may be up-regulated in such conditions. Here, by using a chronic inflammation model, namely subplantar injection of Complete Freund's adjuvant, we show a peripheral synergistic interaction between the histamine H(3) receptor agonist R-(alpha)-methylhistamine and fentanyl on the inhibition of thermal hyperalgesia and of peripheral substance P accumulation. Firstly, dose-related effects obtained for the subplantar antinociceptive effect of fentanyl (0.05-1 microg) in the presence of a fixed dose of R-(alpha)-methylhistamine (12.5 microg) showed a shift to the left when compared to that obtained with fentanyl alone. In a similar way, the subcutaneous administration of fentanyl (0.005-0.1mg/kg) plus a fixed dose of R-(alpha)-methylhistamine (0.5mg/kg) induced a supra additive effect on the inhibition of substance P accumulation in the hind-paw skin of inflamed mice. Interestingly, when a neurokinin-1 receptor antagonist was co-administered, the antinociceptive effects of the combined treatment were potentiated. The peripheral adjuvant effect of R-(alpha)-methylhistamine on fentanyl antinociception and inhibition of substance P accumulation was also demonstrated by means of opioid and histamine H(3) receptors selective antagonists: first, naloxone blockade of fentanyl-mediated effects were partially reversed by co-administration of R-(alpha)-methylhistamine, and second, thioperamide partially antagonised the combined R-(alpha)-methylhistamine/fentanyl effects. Overall, our results clearly show that R-(alpha)-methylhistamine enhances fentanyl effects at peripheral sites, and that the control of substance P levels might be one of the mechanisms responsible of such interaction.

Several motifs found in the third intracellular loop of the M(3) muscarinic receptor are critical for G protein activation and scaffold protein interaction. However, how multiprotein complexes form is not fully understood. A minigene encoding the third intracellular loop of the M(3) muscarinic receptor was constructed to explore whether peptides from this intracellular region could act as inhibitors of the muscarinic multiprotein complex formation and signaling. We found that this construct, when co-expressed with the M(3) receptor, has the ability to act as a competitive antagonist of G protein receptors and receptor-scaffold/accessory proteins. Transient transfection of human embryonic kidney-293 cells with DNA encoding the human M(3) and M(5) receptor subtypes results in a carbachol-dependent increase of inositol phosphate. Co-expression of the M(3) third cytoplasmic loop minigene dramatically reduces both carbachol-mediated G protein activation and inositol phosphate accumulation. Minigene expression also abrogates activation of M(3) and M(5) receptor mitogen-activated protein kinases pathway. Furthermore, minigene expression led to reduced AKT activation. These data, together with results of co-immunoprecipitation of different scaffold and kinase proteins, provide experimental evidence for the role for the third cytoplasmic loop of the human M(3) muscarinic receptor in G-protein activation and multiprotein complex formation.

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.

At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca(2+) influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca(2+) ionophore ionomycin, suggesting a mechanism that is independent of Ca(2+) channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function.

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Adenosine is a well known neuromodulator in the central nervous system. As a consequence, adenosine can be beneficial in certain disorders and adenosine receptors will be potential targets for therapy in a variety of diseases. Adenosine receptors are G protein-coupled receptors, and are also expressed in a large variety of cells and tissues. Using these receptors as a paradigm of G protein-coupled receptors, the present review focus on how protein–protein interactions might contribute to neurotransmitter/neuromodulator regulation, based on the fact that accessory proteins impinge on the receptor/G protein interaction and therefore modulate receptor functioning. Besides affecting receptor signaling, these accessory components also play a key role in receptor trafficking, internalization and desensitization, as it will be reviewed here. In conclusion, the finding of an increasing number of adenosine receptors interacting proteins, and specially the molecular and functional integration of these accessory proteins into receptorsomes, will open new perspectives in the understanding of particular disorders where these receptors have been proved to be involved.

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.