2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

ADP receptors--targets for developing antithrombotic agents

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.     

Preactivation permits subsequent stimulation of phospholipase C by G(i)-coupled receptors

In the complex signal transduction networks involving G protein-coupled receptors there are numerous examples where G(i)-linked receptors augment G(q)-dependent signals. The mechanistic basis of such occurrences is thought to entail signal convergence at phospholipase Cbeta (PLCbeta) via the G protein betagamma-dimers. Herein, we explored the possibility that augmentation by betagamma-dimers requires preactivation of PLCbeta. COS-7 cells were transiently cotransfected with cDNAs encoding various combinations of receptors and G protein subunits. The G(i)-coupled delta- and kappa-opioid receptors could not stimulate PLCbeta unless they were coexpressed with Galpha(16). The opioid-induced response was dose-dependent and partially inhibited by pertussis toxin or coexpression with transducin, indicating the involvement of betagamma-subunits released from the G(i) proteins. When PLCbeta was preactivated by constitutively active mutants of Galpha(16), Galpha(q), or Galpha(14), opioids enhanced the activity by 80 to 300% and such responses were mostly pertussis toxin-sensitive. The opioid-induced enhancement was dose-dependent and could not be blocked by staurosporin, a protein kinase C inhibitor. Other G(i)-coupled receptors that were ineffective on their own also acquired the ability to stimulate PLCbeta in the presence of a constitutively active mutant of Galpha(q). Coactivation of endogenous or exogenous G(q)-coupled receptors with the delta-opioid receptor produced strong stimulations of PLCbeta and such responses could be partially blocked by pertussis toxin. These results show that enhancement of G(q)-dependent signals by G(i)-coupled receptors requires activated PLCbeta and is mediated via the betagamma-dimer.     

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.     

Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y(13) receptor

The human P2Y(13) receptor is a new receptor characterized by coupling to Gi, responsiveness to adenine di-phospho-nucleotides and blockade by the P2Y antagonist AR-C69931MX. The mouse P2Y(13) ortholog has also been reported. Here we report, for the first time, the cloning of rat P2Y(13) receptor, its pharmacological analysis and tissue distribution. Rat P2Y(13) is 79% and 87% identical to human and mouse P2Y(13) receptors, respectively. Expression of rP2Y(13) receptor in 1321N1 cells induced the appearance of responses to the typical P2Y(13) receptor agonists ADP and 2MeSADP, as detected by stimulation of [(35)S]GTPgammaS binding. Agonist activities were higher in cells transfected with rP2Y(13) receptor in the presence of the Galpha(16) subunit; in all cases agonist effects were abolished by pertussis toxin pre-treatment. At variance from both human and mouse receptors, ADP was more potent than 2MeSADP. Other nucleotides and sugar-nucleotides were ineffective. Both in the absence and presence of Galpha(16), activation of rP2Y(13) receptor by ADP and 2MeSADP was completely inhibited by nM concentrations of AR-C69931MX. In contrast, no inhibition of rP2Y(13) receptor was induced by the selective P2Y(1) receptor antagonist MRS2179. rP2Y(13) receptor showed highest expression levels in spleen, followed by liver and brain (with particularly high levels in cortex and striatum as reported in man), suggesting important roles in the nervous and immune systems. Expression levels comparable to those of the other cloned P2Y receptors were found in primary rat astrocytes, indicating a possible role in reactive astrogliosis. Hence, rat P2Y(13) receptor displays several similarities but also interesting differences with its human and mouse orthologs, that will have to be taken into account when characterizing the pathophysiological roles of this receptor in the rat animal models.     

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has approximately 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P(103) in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants (P(103)Y, G(105)D and the double mutant, P(103)Y/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and P(103)Y/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR.     

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

ADP receptor antagonists as antiplatelet therapeutics

With the cloning of the P2Y12 receptor, the molecular basis for ADP-induced platelet aggregation is seemingly complete. Two platelet-bound ADP receptors, P2Y1 and P2Y12, operate through unique pathways to induce and sustain platelet aggregation via the glycoprotein (GP)IIb-IIIa integrin. P2Y1 operates via a glycoprotein q (Gq) pathway, activates phospholipase C, induces platelet shape change and is responsible for intracellular calcium mobilisation. P2Y12 inhibits adenylyl cyclase through a glycoprotein i (Gi)-dependent pathway, and is the target of the clinically used thienopyridines, ticlopidine (Ticlid, F. Hoffman-La Roche) and clopidogrel (Plavix, Bristol-Myers Squibb/Sanofi-Synthelabo). In addition, the receptor is targeted by the ADP analogue AR-C66096, which is currently in Phase IIb clinical trials, as well as other non-nucleoside-based preclinical leads.     

Comparison of the activation kinetics of the m3 acetylcholine receptor and a constitutively active mutant receptor in living cells

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3)-AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.     

Pharmacological characterization of the human P2Y13 receptor

The P2Y13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y12 receptor as well as similar functional properties: coupling to Gi and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y13 receptor and its differences with that of the P2Y12 receptor have been further characterized in 1321N1 cells (binding of [33P]2-methylthio-ADP (2MeSADP) and of GTPgamma[35S]), 1321N1 cells coexpressing Galpha16 [AG32 cells: inositol trisphosphate (IP3) measurement, binding of GTPgamma[35S]) and Chinese hamster ovary (CHO)-K1 cells (cAMP assay)]. 2MeSADP was more potent than ADP in displacing [33P]2MeSADP bound to 1321N1 cells and increasing GTPgamma[35S] binding to membranes prepared from the same cells. Similarly, 2MeSADP was more potent than ADP in stimulating IP3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS-2179) (up to 100 microM). The most potent antagonist was N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (ARC69931MX) (IC50 = 4 nM), which behaved in a noncompetitive way. The active metabolite of clopidogrel was unable to displace bound 2MeSADP at concentrations up to 2 microM.     

Modulation of ligand responses by coupling of alpha(2A)-adrenoceptors to diverse G(alpha)-proteins

The hypothesis that different signalling may be mediated via a single alpha(2A)-adrenoceptor (alpha(2A) AR) subtype was investigated by challenging alpha(2) AR ligands in combination with diverse recombinant wt, mutant, and chimeric G(alpha)-proteins. Possible coupling of alpha(2A) AR to endogenous G(alphai/o)-proteins in CHO-K1 cells was excluded by measuring pertussis toxin (PTX)-resistant [(35)S]GTPgammaS-binding responses as a common functional response to alpha(2A) AR activation. (-)-Adrenaline (10 microM) displayed the highest magnitude of [(35)S]GTPgammaS-binding response in the co-presence of a PTX-resistant G(alphao)Cys(351)Ile protein, whereas a decreased response was obtained with the mutant G(alphai1/2)-proteins. Replacement of the last six amino acids at the C-terminal portion of the G(alphao)-protein by the corresponding amino acid region of either the G(alphaz)-, G(alphas)-, G(alphaq)-, or G(alpha15)-protein and co-expression with the alpha(2A) AR resulted in similar maximal (-)-adrenaline-mediated [(35)S]GTPgammaS-binding responses with these chimeric G(alphao)-proteins. The ligands D-medetomidine, BHT 920 (6-allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-2-ylamine) and (+)-RX 811059 (2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole) were weakly active or virtually inactive at the chimeric G(alphao/s)-, G(alphao/q)-, and G(alphao/15)-proteins in contrast to the G(alphao/z)-protein. Furthermore, combining the constitutively active mutant Thr(373)Lys alpha(2A) AR with these chimeric G(alphao)-proteins enhanced the apparent intrinsic activity of d-medetomidine and BHT 920. A similar observation was made using the corresponding fusion proteins, where the stoichiometry of the mutant alpha(2A) AR to the chimeric G(alphao)-protein was fixed at 1.0. These data indicate that a single ligand may display different magnitudes of activation at the alpha(2A) AR subtype coupled to chimeric G(alphao) proteins under controlled conditions of alpha(2A) AR: G(alphao)-protein expression.     

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y(12) and P2Y(1) inhibition and P2Y(12) and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both G alpha(q) and Galpha (i) signalling. Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y(12) antagonist; MRS2179, a reversible P2Y(1) antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active alpha (IIb)beta(3) (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel. A synergistic effect regarding inhibition of ADP-induced platelet activation (10 microM) was obtained with different combinations of AR-C69931MX and MRS2179. Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect. To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described. Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies.     

Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution

A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.     

Point mutations in either subunit of the GABAB receptor confer constitutive activity to the heterodimer

The GABA receptor (GABABR) is a class C G protein-coupled receptor (GPCR) that functions as an obligate heterodimer, composed of two heptahelical subunits, GABABR subunit 1 (R1) and GABABR subunit 2 (R2). In this study, we generated and pharmacologically characterized constitutively active GABABR mutants as novel tools to explore the molecular mechanisms underlying receptor function. A single amino acid substitution, T290K, in the R1 agonist binding domain results in ligand-independent signaling when this mutant subunit is coexpressed with wild-type R2. Introduction of a Y690V mutation in the putative G protein-coupling domain of R2 is sufficient to confer moderate constitutive activity when this subunit is expressed alone. Activity of the Y690V mutant can be markedly enhanced with coexpression of wild-type R1. Coexpression of both mutant subunits (R1-T290K and R2-Y690K) leads to a further increase in basal signaling. Potencies of the full agonists R-(+)-beta-(aminomethyl)-4-chlorobenzenepropanoic acid hydrochloride (baclofen) and GABA are increased at the constitutively active versus the corresponding wild-type receptors. The mutant GABABR variants provided a sensitive probe enabling detection of inverse or partial agonist activity of molecules previously considered neutral antagonists. Our studies using constitutively active isoforms provide independent support for a model of GABABR function that takes into account 1) ligand binding by R1, 2) signal transduction by R2, and 3) modulation of R2-induced function by R1. Furthermore, we demonstrate that certain hallmark features of constitutive activity as originally established with class A GPCRs (e.g., enhanced agonist potency and affinity), are more generally applicable, as suggested by our finding with a class C heterodimeric receptor.     

Molecular recognition at purine and pyrimidine nucleotide (P2) receptors

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X(1) (derivatives of pyridoxal phosphates and suramin), P2X(3)(A-317491), P2X(7) (derivatives of the isoquinoline KN-62), P2Y(1)(nucleotide analogues MRS 2179 and MRS 2279), P2Y(2)(thiouracil derivatives such as AR-C126313), and P2Y(12)(nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y(1)receptors. The dinucleotide INS 37217 potently activates the P2Y(2)receptor. UTP-gamma-S and UDP-beta-S are selective agonists for P2Y(2)/P2Y(4)and P2Y(6)receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y(1)receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7.     

A "locked-on," constitutively active mutant of the adenosine A1 receptor

We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or "constitutively" active in a [35S]GTPgammaS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar "locked-on" phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPgammaS and the cAMP assay, unlike the wild-type and the two other mutant receptors.     

Generation and analysis of constitutively active and physically destabilized mutants of the human beta(1)-adrenoceptor

Constitutive activity of wild-type and mutant forms of human beta(1)- and beta(2)-adrenoceptors was measured by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays using fusion proteins between these receptors and G(s)alpha. Constitutive activity of the beta(1)-adrenoceptor is enhanced by mutation of Leu(322). The ability of ligands to suppress receptor instability and produce up-regulation is often associated with constitutively active mutants. Leu(322)Lysbeta(1)-adrenoceptor, but not wild type, was up-regulated by exposure to the beta(1)-adrenoceptor selective blocker betaxolol. More extensive sequence alterations of the beta(1)-adrenoceptor were generated to mimic the initially described constitutively active mutant (CAM) of the beta(2)-adrenoceptor that is up-regulated strongly by betaxolol. Substitution of amino acids 316 to 324 of the beta(1)-adrenoceptor with the equivalent alpha(1b)-adrenoceptor sequence did not result in up-regulation by betaxolol. However, these forms of both beta(1)- and beta(2)-adrenoceptors displayed substantial and equivalent constitutive activity. The addition of the Leu(322)Lys mutation into the alpha(1b)-adrenoceptor substituted beta(1)-adrenoceptor to produce the CAMKbeta(1)-adrenoceptor allowed substantially greater levels of up-regulation by betaxolol without enhancement of constitutive [(35)S]GTPgammaS binding. Arg(156)Alabeta(1)-adrenoceptor was up-regulated strongly by betaxolol but displayed lower constitutive activity than did other mutants. Binding of [(35)S]GTPgammaS binding to all the fusion proteins was increased substantially by isoprenaline. Despite the ability of betaxolol to cause up-regulation of many mutants, only for the CAMbeta(2)-adrenoceptor-G(s)alpha and CAMKbeta(1)-adrenoceptor-G(s)alpha fusion proteins was the basal binding of [(35)S]GTPgammaS decreased by betaxolol. Clear resolution between receptor constitutive activity and ligand suppression of receptor instability can be obtained for mutant beta-adrenoceptors, and potential inverse agonists do not function equally at phenotypically apparently equivalent CAM receptors.     

δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors

AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake.     

Random mutagenesis of the human adenosine A2B receptor followed by growth selection in yeast. Identification of constitutively active and gain of function mutations

To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated receptors was determined. All these mutated receptors displayed constitutive activity. In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity. In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active. The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog. Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively. In fact, all constitutively active mutants had an increased potency for NECA. This suggests that the wild-type (wt) human A2B receptor itself is rather silent, which may explain the low affinity of agonists for this receptor. To verify the ability of the mutant receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A receptors. These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt receptor were 0.15 +/- 0.04 and 1.3 +/- 0.4 microM, respectively.     

The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism.