Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

Synthesis and characterization of chiral 6-azaspiro[2.5]octanes as potent and selective antagonists of the M4 muscarinic acetylcholine receptor.

In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.

Gene-edited MLE-15 Cells as a Model for the Hermansky-Pudlak Syndromes.

Defining the mechanisms of cellular pathogenesis in rare lung diseases such as Hermansky-Pudlak syndrome (HPS) is often complicated by loss of the differentiated phenotype of cultured primary alveolar type 2 (AT2) cells, as well as by a lack of durable cell lines that are faithful to both AT2-cell and rare disease phenotypes. We used CRISPR/Cas9 gene editing to generate a series of HPS-specific mutations in the MLE-15 cell line. The resulting MLE-15/HPS cell lines exhibit preservation of AT2 cellular functions, including formation of lamellar body-like organelles, complete processing of surfactant protein B, and known features of HPS specific to each trafficking complex, including loss of protein targeting to lamellar bodies. MLE-15/HPS1 and MLE-15/HPS2 (with a mutation in Ap3ß1) express increased macrophage chemotactic protein-1, a well-described mediator of alveolitis in patients with HPS and in mouse models. We show that MLE-15/HPS9 and pallid AT2 cells (with a mutation in Bloc1s6) also express increased macrophage chemotactic protein-1, suggesting that mice and humans with BLOC-1 mutations may also be susceptible to alveolitis. In addition to providing a flexible platform to examine the role of HPS-specific mutations in trafficking AT2 cells, MLE-15/HPS cell lines provide a durable resource for high-throughput screening and studies of cellular pathophysiology that are likely to accelerate progress toward developing novel therapies for this rare lung disease.

Structural analyses of Candida albicans sterol 14α-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis.

With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as Candida albicans has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of C. albicans CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: (R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of C. albicans CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against Candida krusei and Candida glabrata, pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.

Apical targeting of the P2Y(4) receptor is directed by hydrophobic and basic residues in the cytoplasmic tail.

The P2Y(4) receptor is selectively targeted to the apical membrane in polarized epithelial cell lines and has been shown to play a key role in intestinal chloride secretion. In this study, we delimit a 23 amino acid sequence within the P2Y(4) receptor C-tail that directs its apical targeting. Using a mutagenesis approach, we found that four hydrophobic residues near the COOH-terminal end of the signal are necessary for apical sorting, whereas two basic residues near the NH(2)-terminal end of the signal are involved to a lesser extent. Interestingly, mutation of the key hydrophobic residues results in a basolateral enrichment of the receptor construct, suggesting that the apical targeting sequence may prevent insertion or disrupt stability of the receptor at the basolateral membrane. The signal is not sequence specific, as an inversion of the 23 amino acid sequence does not disrupt apical targeting. We also show that the apical targeting sequence is an autonomous signal and is capable of redistributing the normally basolateral P2Y(12) receptor, suggesting that the apical signal is dominant over the basolateral signal in the main body of the P2Y(12) receptor. The targeting sequence is unique to the P2Y(4) receptor, and sequence alignments of the COOH-terminal tail of mammalian orthologs reveal that the hydrophobic residues in the targeting signal are highly conserved. These data define the novel apical sorting signal of the P2Y(4) receptor, which may represent a common mechanism for trafficking of epithelial transmembrane proteins.

Is GPR17 a P2Y/leukotriene receptor? examination of uracil nucleotides, nucleotide sugars, and cysteinyl leukotrienes as agonists of GPR17.

The orphan receptor GPR17 has been reported to be activated by UDP, UDP-sugars, and cysteinyl leukotrienes, and coupled to intracellular Ca(2+) mobilization and inhibition of cAMP accumulation, but other studies have reported either a different agonist profile or lack of agonist activity altogether. To determine if GPR17 is activated by uracil nucleotides and leukotrienes, the hemagglutinin-tagged receptor was expressed in five different cell lines and the signaling properties of the receptor were investigated. In C6, 1321N1, or Chinese hamster ovary (CHO) cells stably expressing GPR17, UDP, UDP-glucose, UDP-galactose, and cysteinyl leukotriene C4 (LTC4) all failed to promote inhibition of forskolin-stimulated cAMP accumulation, whereas both UDP and UDP-glucose promoted marked inhibition (>80%) of forskolin-stimulated cAMP accumulation in C6 and CHO cells expressing the P2Y14 receptor. Likewise, none of these compounds promoted accumulation of inositol phosphates in COS-7 or human embryonic kidney 293 cells transiently transfected with GPR17 alone or cotransfected with Gαq/i5, which links Gi-coupled receptors to the Gq-regulated phospholipase C (PLC) signaling pathway, or PLCε, which is activated by the Gα12/13 signaling pathway. Moreover, none of these compounds promoted internalization of GPR17 in 1321N1-GPR17 cells. Consistent with previous reports, coexpression experiments of GPR17 with cysteinyl leukotriene receptor 1 (CysLTR1) suggested that GPR17 acts as a negative regulator of CysLTR1. Taken together, these data suggest that UDP, UDP-glucose, UDP-galactose, and LTC4 are not the cognate ligands of GPR17.

Charged residues in the C-terminus of the P2Y1 receptor constitute a basolateral-sorting signal.

The P2Y(1) receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y(1) receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or di-hydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist.

Herein, we report the SAR leading to the discovery of VU6028418, a potent M4 mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.

Discovery of the First Selective M4 Muscarinic Acetylcholine Receptor Antagonists with in Vivo Antiparkinsonian and Antidystonic Efficacy.

Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of nonselective antimuscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here, we utilize genetic mAChR knockout animals in combination with nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.

Synthesis and structure-activity relationships of uracil nucleotide derivatives and analogues as agonists at human P2Y2, P2Y4, and P2Y6 receptors.

A series of UTP, UDP, and UMP derivatives and analogues were synthesized and evaluated at the human pyrimidinergic P2Y receptor subtypes P2Y2, P2Y4, and P2Y6 stably expressed in 1321N1 astrocytoma cells. Substituents at N3 of UTP were poorly tolerated by P2Y2 and P2Y4 receptors. In contrast, a large phenacyl substituent at N3 of UDP was well tolerated by the P2Y6 receptor, yielding a potent and selective P2Y6 receptor agonist (3-phenacyl-UDP, EC50=70 nM, >500-fold selective). The most potent and selective P2Y2 receptor agonist of the present series was 2-thio-UTP (EC50=50 nM, >or=30-fold selective vs P2Y4 and P2Y6). All modifications at the uracil base of UTP led to a decrease in potency at the P2Y4 receptor. A beta,gamma-dichloromethylene modification in the triphosphate chain of 5-bromo-UTP was tolerated by all three receptor subtypes, thus opening up a new strategy to obtain ectonucleotide diphosphohydrolase- and phosphatase-resistant P2Y2, P2Y4, and P2Y6 receptor agonists.

Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome.

Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-ß production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-ß in myeloid cells or of TGF-ß signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-ß, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling.

PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions.

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7(+) endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor-induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

Structural modifications of UMP, UDP, and UTP leading to subtype-selective agonists for P2Y2, P2Y4, and P2Y6 receptors.

A large series of derivatives and analogues of the uracil nucleotides UMP, UDP, and UTP with modifications in various positions of the uracil moiety and/or the phosphate groups were synthesized and evaluated at human P2Y(2), P2Y(4), and P2Y(6) receptors. 2-(Ar)alkylthio substitution of UMP and UDP was best tolerated by the P2Y(2) receptor. 2-Phenethylthio-UMP (13e) showed an EC(50) value of 1.3 µM at P2Y(2) and >70-fold selectivity versus P2Y(4) and P2Y(6) receptors. Substitution of the 2-keto group in UMP by NH (13g, iso-CMP) resulted in the first potent and selective P2Y(4) agonist (EC(50) 4.98 µM, >20-fold selective vs P2Y(2) and P2Y(6)). In contrast, replacement of the 2-keto function in UDP by NH yielded a potent P2Y(2) agonist (12g, iso-CDP, EC(50) = 0.604 µM, >100-fold selective). In an attempt to obtain metabolically stable UTP analogues, ß,γ-dichloro- and ß,γ-difluoro-methylene-UTP derivatives were synthesized. The triphosphate modifications were much better tolerated by P2Y(2), and in some cases also by P2Y(6), than by P2Y(4) receptors. 4-Thio-ß,γ-difluoromethylene-UTP (14g) was a potent P2Y(2) agonist with an EC(50) value of 0.134 µM and >50-fold selectivity. N3-Phenacyl-ß,γ-dichloromethylene-UTP (14b) proved to be a potent P2Y(6) receptor agonist (EC(50) 0.142 µM) with high selectivity versus P2Y(4) (50-fold) and moderate selectivity versus P2Y(2) receptors (6-fold).

Ser352 and Ser354 in the carboxyl terminus of the human P2Y(1) receptor are required for agonist-promoted phosphorylation and internalization in MDCK cells.

BACKGROUND AND PURPOSE: The P2Y(1) receptor promotes chloride secretion in epithelial cells, a process critical for regulation of extracellular ion and fluid levels. Here we have examined the role of phosphorylation in agonist-induced internalization of P2Y(1) receptors. EXPERIMENTAL APPROACH: A high-affinity radiolabelled antagonist, MRS2500, was used to quantify cell surface-binding sites of P2Y(1) receptors in Madin-Darby canine kidney (MDCK) epithelial cells, following exposure to agonists. The regions in the carboxyl terminus involved in both agonist-induced internalization of the receptor and its phosphorylation were identified by mutational analysis. KEY RESULTS: Endogenous and stably expressed recombinant P2Y(1) receptors rapidly internalized with similar time courses in response to agonist in MDCK cells, ensuring that the levels of recombinant receptor achieved by retroviral infection did not adversely affect function of the internalization machinery. Four protein kinase C inhibitors of varying specificity did not affect internalization of recombinant receptors. Agonist-promoted internalization of a series of truncated P2Y(1) receptors identified a region between residues 349 and 359 in the carboxyl terminus as critical for regulation. Two amino acids within this region, Ser352 and Ser354, were shown to be both necessary and sufficient for agonist-promoted receptor phosphorylation and internalization. CONCLUSIONS AND IMPLICATIONS: Our results firmly establish Ser352 and Ser354 in the carboxyl terminus of P2Y(1) receptors as critical residues for agonist-induced receptor internalization in MDCK cells. As the mechanism mediating this regulation requires phosphorylation of these key residues, the relevant receptor-regulated protein kinase can now be identified.

Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation.

Diadenosine 5',5'''-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 microm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

The apical targeting signal of the P2Y2 receptor is located in its first extracellular loop.

P2Y2 and P2Y4 receptors, which have 52% sequence identity, are both expressed at the apical membrane of Madin-Darby canine kidney cells, but the locations of their apical targeting signals are distinctly different. The targeting signal of the P2Y2 receptor is located between the N terminus and 7TM, whereas that of the P2Y4 receptor is present in its C-terminal tail. To identify the apical targeting signal in the P2Y2 receptor, regions of the P2Y2 receptor were progressively substituted with the corresponding regions of the P2Y4 receptor lacking its targeting signal. Characterization of these chimeras and subsequent mutational analysis revealed that four amino acids (Arg95, Gly96, Asp97, and Leu108) in the first extracellular loop play a major role in apical targeting of the P2Y2 receptor. Mutation of RGD to RGE had no effect on P2Y2 receptor targeting, indicating that receptor-integrin interactions are not involved in apical targeting. P2Y2 receptor mutants were localized in a similar manner in Caco-2 colon epithelial cells. This is the first identification of an extracellular protein-based targeting signal in a seven-transmembrane receptor.

Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon.

Eight human G protein-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(12), P2Y(13), and P2Y(14)) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I(sc)) or Ca(2+) mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y(1), P2Y(11), P2Y(12), and P2Y(14) receptors reside at the basolateral membrane, P2Y(2), P2Y(4), and P2Y(6) receptors are expressed at the apical membrane, and the P2Y(13) receptor is unsorted. Biotinylation studies and I(sc) measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the G(q)-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(11)) in lung and colonic epithelial cells (16HBE14o- and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells.

GPR80/99, proposed to be the P2Y(15) receptor activated by adenosine and AMP, is not a P2Y receptor.

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y(15) receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca(2+) mobilization (Inbe et al. J Biol Chem 2004; 279: 19790-9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A(2A) and A(2B) adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188-93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, alpha-ketoglutarate. Consistent with this report, alpha-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts alpha-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by alpha-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.

Agonist versus antagonist action of ATP at the P2Y4 receptor is determined by the second extracellular loop.

UTP is a potent full agonist at both the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor. In contrast, ATP is a potent full agonist at the rP2Y(4) receptor but is a similarly potent competitive antagonist at the hP2Y(4) receptor. To delineate the structural determinants of agonism versus antagonism in these species homologues, we expressed a series of human/rat P2Y(4) receptor chimeras in 1321N1 human astrocytoma cells and assessed the capacity of ATP and UTP to mobilize intracellular Ca(2+). Replacement of the NH(2) terminus of the hP2Y(4) receptor with the corresponding region of the rP2Y(4) receptor resulted in a receptor that was activated weakly by ATP, whereas replacement of the second extracellular loop (EL2) of the hP2Y(4) receptor with that of the rP2Y(4) receptor yielded a chimeric receptor that was activated fully by UTP and near fully by ATP, albeit with lower potencies than those observed at the rP2Y(4) receptor. These potencies were increased, and ATP was converted to a full agonist by replacing both the NH(2) terminus and EL2 in the hP2Y(4) receptor with the corresponding regions from the rP2Y(4) receptor. Mutational analysis of the five divergent amino acids in EL2 between the two receptors revealed that three amino acids, Asn-177, Ile-183, and Leu-190, contribute to the capacity of EL2 to impart ATP agonism. Taken together, these results suggest that the second extracellular loop and the NH(2) terminus form a functional motif that plays a key role in determining whether ATP functions as an agonist or antagonist at mammalian P2Y(4) receptors.