Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases.

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), and diadenosine 5',5"'-P1,P5-pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 micro m, 8.0 micro m, and 49.5 micro m for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.     

Isothermal titration calorimetry reveals a zinc ion as an atomic switch in the diadenosine polyphosphates

Diadenosine polyphosphates (diadenosine 5',5'-P(1),P(n)-polyphosphate (Ap(n)A)) are 5'-5'-phosphate-bridged dinucleosides that have been proposed to act as signaling molecules in a variety of biological systems. Isothermal titration calorimetry was used to measure the affinities of a variety of metal cations for ATP, diadenosine 5',5'-P(1),P(3)-triphosphate (Ap(3)A), diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A), and diadenosine 5',5'-P(1),P(5)-pentaphosphate (Ap(5)A). The binding of Mg(2+), Ca(2+), and Mn(2+) to ATP is shown to take place with the beta,gamma-phosphates (primary site) and be endothermic in character. The binding of Ni(2+), Cd(2+), and Zn(2+) to ATP is found to take place at both the primary site and at a secondary site identified as N-7 of the adenine ring. Binding to this second site is exothermic in character. Generally, the binding of metal cations to diadenosine polyphosphates involves a similar primary site to ATP. No exothermic binding events are identified. Critically, the binding of Zn(2+) to diadenosine polyphosphates proves to be exceptional. This appears to involve a very high affinity association involving the N-7 atoms of both adenine rings in each Ap(n)A, as well as the more usual endothermic association with the phosphate chain. The high affinity association is also endothermic in character. A combination of NMR and CD evidence is provided in support of the calorimetry data demonstrating chemical shift changes and base stacking disruptions entirely consistent with N-7 bridging interactions. N-7 bridging interactions are entirely reversible, as demonstrated by EDTA titration. Considering the effects of Zn(2+) on a wide variety of dinucleoside polyphosphate-metabolizing enzymes, we examine the possibility of Zn(2+) acting as an atomic switch to control the biological function of the diadenosine polyphosphates.     

High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.     

Yeast diadenosine 5',5'-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase

The diadenosine 5',5'-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.     

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.     

Isolation and characterization of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum

A new enzyme that hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate has been purified by a factor of 250 from the acellular slime mold Physarum polycephalum. Activity was assayed radioisotopically with [3H]Ap4A. Isolation of the enzyme was facilitated by dye-ligand chromatography. The enzyme symmetrically hydrolyzes Ap4A to ADP and exhibits biphasic kinetics for the substrate with values for the apparent Km of 2.6 micro M and 37 micro M. The two values of Vmax differ by a factor of 10. Mg2+, Ca2+, and other divalent cations inhibit the activity with 40-80% inhibition occurring at 0.5 mM. Mg2+, at 0.5 mM, decreases both values of Vmax by 50%, decreases the low Km value by about 30%, and increases the high Km value by about 100%. (Ethylenedinitrilo)tetraacetic acid (EDTA) and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), at 10 mM, inhibit the activity by 50%. ADP, ATP, Ap4, and Gp4 are equipotent inhibitors with 50% inhibition occurring at 30 micro M. AMP is a relatively weak inhibitor. The molecular weight of the enzyme is 26000 on the basis of elution of activity from a calibrated Sephadex G-75 column.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Specific magnesium-dependent diadenosine 5'',5''''''-P1,P3-triphosphate pyrophosphohydrolase in Escherichia coli.

A specific Mg2+-dependent bis(5'-adenosyl)-triphosphatase (EC 3.6.1.29) was purified 270-fold from Escherichia coli. The enzyme had a strict requirement for Mg2+. Other divalent cations, such as Mn2+, Ca2+, or Co2+, were not effective. The products of the reaction with bis(5'-adenosyl) triphosphate (Ap3A) as the substrate were ADP and AMP in stoichiometric amounts. The Km for Ap3A was 12 +/- 5 microM. Bis(5'-adenosyl) di-, tetra-, and pentaphosphates, NAD+, ATP, ADP, AMP, glucose 6-phosphate, p-nitrophenylphosphate, bis-p-nitrophenylphospate, and deoxyribosylthymine-5'-(4-nitrophenylphosphate) were not substrates of the reaction. The enzyme had a molecular mass of 36 kilodaltons (as determined both by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of 4.84 +/- 0.05, and a pH optimum of 8.2 to 8.5. Zn2+, a known potent inhibitor of rat liver bis(5'-adenosyl)-triphosphatase and bis(5'-guanosyl)-tetraphosphatase (EC 3.6 1.17), was without effect. The enzyme differs from the E. coli diadenosine 5',5'-P1, P4-tetraphosphate pyrophosphohydrolase which, in the presence of Mn2+, also hydrolyzes Ap3A.     

Diadenosine 5', 5"'-P1,P4-tetraphosphate stimulates processing of adp-ribosylated poly(ADP-ribose) polymerase

The effect of diadenosine 5', 5"'-P1,P4-tetraphosphate (Ap4A) on the time course and acceptors of poly(ADP-ribose) synthesis was studied in undamaged and N-methyl-N'-nitro-N-nitrosoguanidine-treated human lymphocytes. Analysis of protein acceptors of poly(ADP-ribose) revealed that treatment with Ap4A stimulated ADP-ribosylation of bands at molecular weights of 96,000, 79,000, and 62,000. Pulse-chase studies showed that these bands were produced as a result of an effect of Ap4A on the processing of ADP-ribosylated proteins rather than on the synthesis of newly ADP-ribosylated proteins. By incubating permeabilized cells in the absence or presence of Ap4A and purified poly(ADP-ribose) polymerase auto-ADP-ribosylated with [32P]NAD+, we showed that the Mr = 96,000, 79,000, and 62,000 bands were derivatives of the prelabeled enzyme. Our results indicate that normal human lymphocytes process auto-ADP-ribosylated poly(ADP-ribose) polymerase to specific lower molecular weight products and that this processing is stimulated by Ap4A.     

Replicon initiation frequency and intracellular levels of ATP, ADP, AMP and of diadenosine 5',5'-P1,P4-tetraphosphate in ehrlich ascites cells cultured aerobically and anaerobically

When Ehrlich ascites cells were cultured for 2 h under oxygen-free atmosphere, a shut-down of initiation of new replication units was observed by chain length analysis of the nascent daughter strands and by DNA fibre autoradiography. The intracellular level of ATP, ADP and AMP remained virtually normal in the anaerobized cells, while that of diadenosine 5',5'-P1,P4-tetraphosphate was found reduced by about two orders of magnitude. It is proposed that the ceasing of DNA synthesis after O2 removal is at actively controlled regulatory response of the cells in which diadenosine 5',5"'-P1,P4-tetraphosphate is probably involved.     

The duality of LysU, a catalyst for both Ap4A and Ap3A formation

Heat shock inducible lysyl-tRNA synthetase of Escherichia coli (LysU) is known to be a highly efficient diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) synthase. However, we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activity, initially producing Ap4A from ATP, before converting that tetraphosphate to a triphosphate. LysU appears to be an effective diadenosine 5',5'-P1,P3-triphosphate (Ap3A) synthase. Mechanistic investigations reveal that Ap3A formation requires: (a) that the second step of Ap4A formation is slightly reversible, thereby leading to a modest reappearance of adenylate intermediate; and (b) that phosphate is present to trap the intermediate (either as inorganic phosphate, as added ADP, or as ADP generated in situ from inorganic phosphate). Ap3A forms readily from Ap4A in the presence of such phosphate-based adenylate traps (via a 'reverse-trap' mechanism). LysU is also clearly demonstrated to exist in a phosphorylated state that is more physically robust as a catalyst of Ap4A formation than the nonphosphorylated state. However, phosphorylated LysU shows only marginally improved catalytic efficiency. We note that Ap3A effects have barely been studied in prokaryotic organisms. By contrast, there is a body of literature that describes Ap3A and Ap4A having substantially different functions in eukaryotic cells. Our data suggest that Ap3A and Ap4A biosynthesis could be linked together through a single prokaryotic dual 'synthase' enzyme. Therefore, in our view there is a need for new research into the effects and impact of Ap3A alone and the intracellular [Ap3A]/[Ap4A] ratio on prokaryotic organisms.     

Analogs of diadenosine tetraphosphate (Ap4A)

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.     

Nuclear AP4A-binding activity of sea urchin embryos changes in relation to the initiation of S phase

The AP4A-binding activity of sea urchin embryos was studied using radioactively labelled diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). Among various subcellular components that can bind [3H]AP4A, nuclei alone showed the highly specific Ap4A-binding activity which was not influenced by the presence of AP4A, AP5A and GP4G. The addition of an excess amount of ATP only slightly reduced the binding of [3H]AP4A to the nuclei. It was found that AP4A binds to the residual proteinaceous structure of nuclei which was resistant to the extraction with 2 M NaCl. The nuclear AP4A-binding activity fluctuated cyclically during each cell cycle, with a transient increase at the beginning of S phase followed by an abrupt decrease within 10 min. When the initiation of S phase was blocked, the increase in the AP4A-binding activity was also prevented. It seems that the binding of AP4A to the nuclear structural protein is involved in the initiation of S phase.     

Regulation of hepatic parenchymal and non-parenchymal cell function by the diadenine nucleotides Ap3A and Ap4A

The diadenine nucleotides diadenosine 5',5"-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A) can be released from platelets and were shown to act as long-lived signal molecules. Accordingly, we studied their potential effect on hepatic metabolism. In isolated perfused rat liver, Ap3A and Ap4A increase the portal pressure, lead to a transient net release of Ca2+, complex net K+ movement across the liver plasma membrane and stimulate hepatic glucose output and 14CO2 production from [1-14C]glutamate. These responses resemble that obtained with extracellular ATP. This and studies on the additivity of ATP and Ap4A effects suggest similar mechanisms mediating the ATP and diadenine nucleotide effects in the liver. Ap3A and Ap4A increased the activity of glycogen phosphorylase a in isolated hepatocyte suspensions by about 100%, pointing to a direct effect of these nucleotides on hepatic parenchymal cells. A response of hepatic non-parenchymal cells to diadenine nucleotide infusion is suggested by a marked stimulation of thromboxane and prostaglandin D2 release from perfused liver. Studies with the thromboxane A2 receptor antagonist BM 13.177 (20 microM) show that the pressure and glucose response to the diadenine nucleotides is partially mediated by this thromboxane formation. Studies with retrograde and sequential liver perfusions suggest a less efficient degradation of the diadenine nucleotides during a single liver passage compared to extracellular ATP. The data suggest that Ap3A and Ap4A are potential regulators of hepatic hemodynamics and metabolism, involving complex interactions between hepatic parenchymal cells and hepatic non-parenchymal cells, including eicosanoids as signal molecules.     

ADP-ribosylation of diadenosine 5',5"-P1,P4-tetraphosphate by poly(ADP-ribose) polymerase in vitro

When the effect of diadenosine 5',5"'-P1,P4-tetraphosphate on a purified poly(ADP-ribose) polymerase reaction was examined, the compound strongly inhibited ADP-ribosylation reaction of histone, while the compound was much less inhibitory of the Mg2+-dependent automodification of this enzyme. In an attempt to study the mechanism of the inhibition, we analyzed the total reaction products, which were synthesized from NAD+ in the presence of diadenosine 5',5"'-P1,P4-tetraphosphate in a reaction mixture for ADP-ribosylation of histone, and found that a new, low molecular product was predominantly synthesized instead of ADP-ribosylated histone in the reaction. Approximately 90% of added NAD+ was converted into this low molecular product under an appropriate reaction condition. Further analysis revealed that the product was mono- and oligo(ADP-ribosyl)ated diadenosine nucleotide and that the bound oligo(ADP-ribose) is elongating at one end of the product during the reaction. Thus, the present study clearly demonstrated that diadenosine 5',5"'-P1,P4-tetraphosphate functions as an acceptor for ADP-ribose in a poly(ADP-ribose) polymerase reaction in vitro. The finding that histone H1 is required in the reaction mixture for the synthesis of this new product suggests that histone H1 and the diadenosine compound interact during this modification reaction.     

A proton magnetic resonance study of the effects of polyamine and divalent metal ions on diadenosine 5',5'-P1,P4-tetraphosphate base stacking

Complexation of putrescine, spermidine, spermine, and Mg2+ with diadenosine 5',5'-P1,P4-tetraphosphate induces an upfield shift in the signals for the H-2 and H-8 protons. The upfield shifts in H-2 indicate that cation complexation enhances intramolecular adenine stacking interactions. The resonances for H-2 and H-8 of neutral analogs of 5',5'-dinucleotides appear farther upfield relative to the appropriate monomeric models than those for the corresponding dinucleotide; reduction of intra-chain phosphate repulsion is the origin of cation induced enhancement of diadenosine 5H,5'-P1,P4-tetraphosphate base stacking.     

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae.

The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by organellar and cytoplasmic phenylalanyl-tRNA synthetases of Euglena gracilis

Purified phenylalanyl-tRNA synthetases present in chloroplasts, mitochondria and cytoplasm of green and bleached Euglena gracilis strains, respectively, are able to synthesize diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A). Ap4A synthesis is strictly dependent on zinc ions. This is the first evidence that chloroplasts should be able to synthesize Ap4A. Synthesis of Ap4A by phenylalanyl-tRNA synthetases of the three compartments of a plant cell or by other enzymes such as Ap4A phosphorylase is discussed.     

Mitochondrial location of rat liver dinucleoside triphosphatase

Rat liver dinucleoside triphosphatase (EC 3.6.1.29) is associated with sucrose-gradient purified mitochondria and can be extracted by freeze and thaw treatment. The proportion of mitochondrial dinucleoside triphosphatase approaches 50% of total liver enzyme. Evidence is also presented that 10% of total liver bis(5'-guanosyl)tetraphosphatase (EC 3.6.1.17) might be equally linked to mitochondria. Those data suggest that diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, or other substrates of those enzymes, might be somehow related to mitochondria or mitochondrial function(s), although the occurrence of dinucleoside polyphosphates has not been reported in that organelle.     

In human and rat lung membranes [35S]GTPgammaS binding is a tool for pharmacological characterization of G protein-coupled dinucleotide receptors.

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y2 receptor subtype plays a role in controlling Cl- and fluid transport. Besides ATP or UTP, also diadenosine tetraphosphate (Ap4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [35S]GTPgammaS binding. Stimulation of [35S]GTPgammaS binding to G proteins has already been successfully applied to elucidate agonist binding to various G protein-coupled receptors. Ap(n)A (n = 2 to 6) enhanced [35S]GTPgammaS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates. Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([3H]Ap4A) binding site. The affinity for Ap2A, Ap3A, Ap4A, Ap5A, and Ap6A was also comparable. 8-Diazido-Ap4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.