Adenosine receptors interacting proteins (ARIPs): Behind the biology of adenosine signaling

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.     

Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors

G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss.     

Adenosine receptor containing oligomers: their role in the control of dopamine and glutamate neurotransmission in the brain

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.     

Synergism of accessory factors in functional expression of mammalian odorant receptors

The discovery of odorant receptors led to endeavors in matching them with their cognate ligands. Although it has been challenging to functionally express odorant receptors in heterologous cells, previous studies have linked efficient odorant receptor expression with N-terminal modifications and accessory proteins, including the receptor-transporting proteins (RTPs) and Ric8b. Here we have shown that a shorter form of RTP1, RTP1S, supports robust cell-surface and functional expression of representative odorant receptors. Using a combination of accessory proteins, including RTP1S, Ric8b, and G(alphaolf), a diverse set of untagged odorant receptors were successfully expressed heterologously due to the synergistic effects among the various accessory proteins. Furthermore, the addition of an N-terminal rhodopsin tag to the odorant receptors, along with the same set of accessory proteins, exhibits an additional level of synergism, inducing enhanced odorant receptor responses to odorants and thus defining a more efficient heterologous expression system. We then showed that the presence or absence of different N-terminal tags has little effect on the ligand specificity of odorant receptors, although the amount of receptor expressed can play a role in the ligand response profile. The accuracy of the odorant receptor heterologous expression system involving tagged odorant receptors and various accessory proteins promises success in high throughput de-orphaning of mammalian odorant receptors.     

A role for G proteins in plant hormone signalling?

The G protein signalling pathway is one of the most highly conserved mechanisms that enables cells to sense and respond to changes in their environment. Essential components of this are cell surface G protein-coupled receptors (GPCRs) that perceive extracellular ligands, and heterotrimeric G proteins (G proteins) that transduce information from activated GPCRs to down-stream effectors such as enzymes or ion channels. It is now clear from a range of biochemical and molecular studies that some potential G protein signalling components exist in plants. The best examples of these are the seven transmembrane receptor homologue GCR1 and the G_α (GPA1) and G_β (Gβ1) subunit homologues of heterotrimeric G proteins. G protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate G proteins in a range of signalling pathways that include the plant hormones gibberellin and auxin. Furthermore, antisense suppression of GCR1 expression in Arabidopsis leads to a phenotype that supports a role for this receptor in cytokinin signalling. This review considers the current evidence for and against functional G protein signalling pathways in higher plants and questions whether or not these might be involved in the action of certain plant hormones.     

Internalization and desensitization of adenosine receptors

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A(1), A(2A), A(2B) and A(3) receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A(1)Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A(2A)R and A(2B)R undergo much faster downregulation, usually shorter than 1 h. The A(3)R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A(3)R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.     

The effects of C-terminal truncation of receptor activity modifying proteins on the induction of amylin receptor phenotype from human CTb receptors

Receptor activity modifying proteins (RAMPs) interact with calcitonin receptors to produce novel amylin receptor phenotypes. We have recently demonstrated that the short intracellular C-terminus of RAMPs plays a key role in the function of amylin receptors derived from the CTa calcitonin receptor through the use of chimeric RAMPs and RAMPs that are truncated at the C-terminus [15, Udawela M, Christopoulos G, Morfis M, Christopoulos A, Ye S, Tilakaratne N, Sexton PM. A critical role for the short intracellular C terminus in receptor activity modifying protein function. Mol Pharmacol 2006;70:1750-60., 18, Udawela M, Christopoulos G, Tilakaratne N, Christopoulos A, Albiston A, Sexton PM. Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors. Mol Pharmacol 2006;69:1984-89.]. The calcitonin receptor in humans is expressed as two major alternatively spliced isoforms termed CTa and CTb. Relatively little is known about how alternate splicing of the receptor affects the interaction between calcitonin receptors and RAMPs. We have examined the effect of RAMP truncation, through use of mutant constructs that delete the last 8 amino acids of each of the 3 known human RAMPs, and characterised these for interaction with CTb receptors through co-expression in COS-7 cells. As seen with the CTa receptor isoform, RAMP truncation caused a marked loss in induction of AMYb receptor phenotypes as characterised by (125)I-rat amylin radioligand binding assays and cAMP accumulation assays; the latter as a marker of receptor signalling. The effect was most pronounced for RAMP1 and RAMP2 deletion mutants, but attenuated responses were also observed with co-expressed RAMP3 deletion mutants. These data support a direct role for the RAMP C-terminus in the interaction of RAMP/calcitonin receptor complexes with intracellular accessory proteins involved in signalling and/or receptor trafficking.     

The calcium-sensing receptor and its interacting proteins

Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling personalities. The extracellular Ca-sensing receptor (CaR) signals via Galpha(i), Galpha(q) and Galpha(12/13), but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling.     

Th1 cytokines regulate adenosine receptors and their downstream signaling elements in human microvascular endothelial cells

We and others have shown that adenosine, acting at its receptors, is a potent modulator of inflammation and angiogenesis. To better understand the regulation of adenosine receptors during these processes we studied the effects of IL-1, TNF-alpha, and IFN-gamma on expression and function of adenosine receptors and select members of their coupling G proteins in human dermal microvascular endothelial cells (HMVEC). HMVEC expressed message and protein for A(2A) and A(2B), but not A(1) or A(3) receptors. IL-1 and TNF-alpha treatment increased message and protein expression of A(2A) and A(2B) receptor. IFN-gamma treatment also increased the expression of A(2B) receptors, but decreased expression of A(2A) receptors. Resting HMVEC and IFN-gamma-treated cells showed minimal cAMP response to the selective A(2A) receptor agonist 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094). In contrast, MRE0094 stimulated a dose-dependent increase in cAMP levels in TNF-alpha-treated cells that was almost completely blocked by the A(2A) receptor antagonist ZM-241385 (4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl]phenol). The nonselective adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine increased cAMP levels in both TNF-alpha- and IFN-gamma-treated cells, but not control cells, and its effect was only partially reversed by ZM-241385 in TNF-alpha-treated cells and not affected in IFN-gamma-treated cells. HMVEC expressed a higher level of G protein beta1 isoform than beta4 isoform. Although none of the cytokines tested affected G(beta1) expression, both IL-1 and TNF-alpha significantly up-regulated G(beta4) expression. These findings indicate that inflammatory cytokines modulate adenosine receptor expression and function on HMVECs and suggest that the interaction between proinflammatory cytokines and adenosine receptors may affect therapeutic responses to anti-inflammatory drugs that act via adenosine-dependent mechanisms.     

Receptor crosstalk. Implications for cardiovascular function, disease and therapy.

There are at least three well-defined signalling cascades engaged directly in the physiological regulation of cardiac circulatory function: the beta1-adrenoceptors that control the cardiac contractile apparatus, the renin-angiotensin-aldosterone system involved in regulating blood pressure and the natriuretic peptides contributing at least to the factors determining circulating volume. Apart from these pathways, other cardiac receptor systems, particularly the alpha1-adrenoceptors, adenosine, endothelin and opioid receptors, whose physiological role may not be immediately evident, are also important with respect to regulating cardiovascular function especially in disease. These and the majority of other cardiovascular receptors identified to date belong to the guanine nucleotide binding (G) protein-coupled receptor families that mediate signalling by coupling primarily to three G proteins, the stimulatory (Gs), inhibitory (Gi) and Gq/11 proteins to stimulate the adenylate cyclases and phospholipases, activating a small but diverse subset of effectors and ion channels. These receptor pathways are engaged in crosstalk utilizing second messengers and protein kinases as checkpoints and hubs for diverting, converging, sieving and directing the G protein-mediated messages resulting in different signalling products. Besides, the heart itself is endowed with the means to harmonize these signalling mechanisms and to fend off potentially fatal consequences of functional loss of the essential signalling pathways via compensatory reserve pathways, or by inducing some adaptive mechanisms to be turned on, if and when required. This receptor crosstalk constitutes the underlying basis for sustaining a coherently functional circulatory entity comprising mechanisms controlling the contractile apparatus, blood pressure and circulating volume, both in normal physiology and in disease.     

Adenosine receptors and membrane microdomains

Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors. The four adenosine receptor subtypes-A(1), A(2a), A(2b), A(3)-exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of G protein coupled receptor signaling at the level of protein-protein interactions as well as through signaling cross talk. With respect to adenosine receptors, the activation of one receptor subtype can have profound direct effects in one cell type but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of G protein coupled receptor signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling.     

Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii

ABSTRACT: BACKGROUND: Adaptive responses in fungi result from the interaction of membrane receptors and extracellular ligands. Many different classes of receptors have been described in eukaryotic cells. Recently a new family of receptors classified as belonging to the progesterone-adiponectin receptor (PAQR) family has been identified. These receptors have the seven transmembrane domains characteristic of G-protein coupled receptors, but their activity has not been associated directly to G proteins. They share sequence similarity to the eubacterial hemolysin III proteins. RESULTS: A new receptor, SsPAQR1 (Sporothrix schenckii progesterone-adiponectinQ receptor1), was identified as interacting with Sporothrix schenckii G protein alpha subunit SSG-2 in a yeast two-hybrid assay. The receptor was identified as a member of the PAQR family. The cDNA sequence revealed a predicted ORF of 1542 bp encoding a 514 amino acids protein with a calculated molecular weight of 57.8 kDa. Protein domain analysis of SsPAQR1 showed the 7 transmembrane domains (TM) characteristic of G protein coupled receptors and the presence of the distinctive motifs that characterize PAQRs. A yeast-based assay specific for PAQRs identified progesterone as the agonist. S. schenckii yeast cells exposed to progesterone (0.50 mM) showed an increase in intracellular levels of 3[PRIME], 5[PRIME] cyclic adenosine monophosphate (cAMP) within the first min of incubation with the hormone. Different progesterone concentrations were tested for their effect on the growth of the fungus. Cultures incubated at 35[DEGREE SIGN]C did not grow at concentrations of progesterone of 0.05 mM or higher. Cultures incubated at 25[DEGREE SIGN]C grew at all concentrations tested (0.01 mM-0.50 mM) with growth decreasing gradually with the increase in progesterone concentration. CONCLUSION: This work describes a receptor associated with a G protein alpha subunit in S. schenckii belonging to the PAQR family. Progesterone was identified as the ligand. Exposure to progesterone increased the levels of cAMP in fungal yeast cells within the first min of incubation suggesting the connection of this receptor to the cAMP signalling pathway. Progesterone inhibited the growth of both the yeast and mycelium forms of the fungus, with the yeast form being the most affected by the hormone.     

Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes"

Many cellular functions are carried out by multiprotein complexes. The last five years of research have revealed that many G-protein coupled receptor (GPCR) functions that are not mediated by G proteins involve protein networks, which interact with their intracellular domains. This review focuses on one family of GPCRs activated by serotonin, the 5-HT(2) receptor family, which comprises three closely related subtypes, the 5-HT(2A), the 5-HT(2B) and the 5-HT(2c) receptors. These receptors still raise particular interest, because a large number of psychoactive drugs including hallucinogens, anti-psychotics, anxiolytics and anti-depressants, mediate their action, at least in part, through activation of 5-HT(2) receptors. Recent studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that the C-terminal domains of 5-HT(2) receptors interact with intracellular proteins. To date, the protein network associated with the C-terminus of the 5-HT(2C) receptor has been the most extensively characterized, using a proteomic approach combining affinity chromatography, mass spectrometry and immunoblotting. It includes scaffolding proteins containing one or several PDZ domains, signalling proteins and proteins of the cytoskeleton. Data indicating that the protein complexes interacting with 5-HT(2) receptor C-termini tightly control receptor trafficking and receptor-mediated signalling will also be reviewed.     

Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach

Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

AGS proteins, GPR motifs and the signals processed by heterotrimeric G proteins

A long term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation by heterotrimeric G-proteins (G). G-proteins play a central role in cellular communication mediating the cell response to numerous hormones and neurotransmitters. A major determinant of signalling specificity for heterotrimeric G-proteins is the cell specific expression of the subtypes of the primary signalling entities, receptor, G and effector (E). Another major site for regulating signalling specificity lies at the R-G or G-E interface where these interactions are influenced by cell architecture, the stoichiometry of signalling components and accessory proteins that may segregate the receptor to microdomains of the cell, regulate the efficiency and/or specificity of signal transfer and/or influence the activation state of G-protein independent of a classical G-protein coupled receptor. One strategy to address these issues in our laboratory involves the identification of cellular proteins that regulate the transfer of signal from receptor to G or directly influence the activation state of G independent of a classical G-protein coupled receptor. We identified three proteins, AGS1, AGS2 and AGS3 (for Activators of G-protein Signaling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1, 2 and 3 interact with different subunits and/or conformations of heterotrimeric G-proteins, selectively activate different G-proteins, provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers.     

Receptor activity-independent recruitment of betaarrestin2 reveals specific signalling modes

The roles of betaarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, betaarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with betaarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the betaarrestin/receptor complexes. Interestingly, betaarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating betaarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of betaarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by betaarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that betaarrestin2 can act as a 'bonafide' signalling molecule even in the absence of activated receptor.     

Wnt3a stimulation elicits G-protein-coupled receptor properties of mammalian Frizzled proteins

Receptors of the Fz (Frizzled) family initiate Wnt ligand-dependent signalling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains, and their signalling depends on heterotrimeric G-proteins in various organisms; however, Fz proteins constitute a distinct group within the GPCR (G-protein-coupled receptor) superfamily, and Fz signalling can be G-protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. In the present study, we demonstrate that mammalian Fz proteins act as GPCRs on heterotrimeric G(o/i) proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine-nucleotide exchange on G(o/i) proteins. These responses were sensitive to a Wnt antagonist and to pertussis toxin, which decouples the G(o/i) proteins from their receptors through covalent modification. The results of the present study provide the long-awaited biochemical proof of the GPCR nature of Fz receptors.     

Dual Signalling by the Adenosine A2a Receptor Involves Activation of Both N- and P-Type Calcium Channels by Different G Proteins and Protein Kinases in the Same Striatal Nerve Terminals

Abstract: Many G_s-linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A_2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of G_s, adenylyl cyclase, protein kinase A, and P-type calcium channels; the other is mediated by a cholera toxin-insensitive G protein, protein kinase C, and N-type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A_2a receptor antagonist KF17837. This demonstrates that the A_2a receptor activates two signalling systems in striatal cholinergic neurons.