Inhibition of adenylyl and guanylyl cyclase isoforms by the antiviral drug foscarnet.

The pyrophosphate (PP(i)) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PP(i) in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by Galpha(s) relieved the inhibition by foscarnet but not that by PP(i). The IC(50) of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines; foscarnet blocked cAMP accumulation after A(2A)-adenosine receptor stimulation in PC12 but not in HEK-A(2A) cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase. Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.

Metal-dependent nucleotide binding to the Escherichia coli rotamase SlyD.

Upon expression and purification of the first catalytic domain of mammalian adenylate cyclase type 1 (IC1), a 27 kDa contaminant was observed, which was labelled by three radioactive ATP analogues (8-azido-ATP, 3'-O-(4-benzoyl)benzoyl-ATP and 2',3'-dialdehyde-ATP); the protein was purified separately and identified as Escherichia coli SlyD by N-terminal amino acid sequence determination. SlyD is the host protein required for lysis of E. coli upon infection with bacteriophage PhiX174 and has recently been shown to display rotamase (peptidylproline cis-trans-isomerase) activity. The covalent incorporation of ATP analogues into SlyD was promoted by bivalent transition metal ions (Zn(2+)>/=Ni(2+)>Co(2+)>Cu(2+)) but not by Mg(2+) or Ca(2+); this is consistent with the known metal ion specificity of SlyD. ATP, ADP, GTP and UTP suppressed labelling of SlyD with comparable potencies. Similarly, SlyD bound 2',3'-O-(-2,4, 6-trinitrophenyl)-ATP with an affinity in the range of 10 microM, as determined by fluorescence enhancement. This interaction was further augmented in the presence of Zn(2+) (K(d)= approximately 2 microM at saturating Zn(2+)) but not of Mg(2+). Irrespective of the assay conditions, hydrolysis of nucleotides by SlyD was not detected. Upon gel filtration on a Superose HR12 column, SlyD (predicted molecular mass=21 kDa) migrated with an apparent molecular mass of 44 kDa, indicating that the protein was a dimer. However, the migration of SlyD was not affected by the presence of Zn(2+) or of Zn(2+) and ATP. Thus we concluded that SlyD binds nucleotides in the presence of metal ions. These findings suggest that SlyD serves a physiological role that goes beyond that accounted for by its intrinsic rotamase activity, which is observed in the absence of metal ions.

The C2 catalytic domain of adenylyl cyclase contains the second metal ion (Mn2+) binding site.

Membrane-bound mammalian adenylyl cyclase isoforms contain two internally homologous cytoplasmic domains (C1 and C2). When expressed separately, C1 and C2 are catalytically inactive, but conversion of ATP to cAMP is observed if C1 and C2 are combined. By analogy with DNA polymerases, adenylyl cyclases are thought to require two divalent metal ions for nucleotide binding and phosphodiester formation; however, only one Mg2+ ion (liganded to C1) has been visualized in the recently solved crystal structure of a C1-C2 complex [Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916]. Here, we have studied the binding of ATP to IIC2 (from type II adenylyl cyclase) using ATP analogues [2',3'-dialdehyde ATP (oATP), a quasi-irreversible inhibitor that is covalently incorporated via reduction of a Schiff base, the photoaffinity ligand 8-azido-ATP (8N3-ATP), and trinitrophenyl-ATP (TNP-ATP), a fluorescent analogue] and fluorescein isothiocyanate (FITC). [alpha-32P]oATP and 8N-[alpha-32P]ATP are specifically incorporated into IIC2. Labeling of IIC2 by [alpha-32P]oATP and by FITC is greatly enhanced by Mn2+ and to a much lesser extent by Mg2+. Similarly, TNP-ATP binds to IIC2 as determined by fluorescence enhancement, and this binding is promoted by Mn2+. Thus, a second metal ion binding site (preferring Mn2+) is contained within the C2 domain, and this finding highlights the analogy in the reaction catalyzed by DNA polymerases and adenylyl cyclases.

Inhibition of receptor/G protein coupling by suramin analogues.

Suramin analogues act as direct antagonists of heterotrimeric G proteins because they block the rate-limiting step of G protein activation (i.e., the dissociation of GDP prebound to the G protein alpha subunit). We have used the human brain A1 adenosine receptor and the rat striatal D2 dopamine receptor, two prototypical Gi/G(o)-coupled receptors, as a model system to test whether the following analogues suppress the receptor-dependent activation of G proteins: 8-(3-nitrobenzamido)-1,3,5-naphthalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)-benzamido)-1,3,5-naphthalenetrisulfonic acid (NF018); 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalenetr isulfonic acid) (NF023); 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1- phenylene)) bis(1,3,5-naphthalenetrisulfonic acid) (NF037); and suramin. Suramin and its analogues inhibit the formation of the agonist-specific ternary complex (agonist/receptor/G protein). This inhibition is (i) quasicompetitive with respect to agonist binding in that it can be overcome by increasing receptor occupancy but (ii) does not result from an interaction of the analogues with the ligand binding pocket of the receptors because the binding of antagonists or of agonists in the absence of functional receptor/G protein interaction is not affected. In addition to suppressing the spontaneous release of GDP from defined G protein alpha subunits, suramin and its analogues reduce receptor-catalyzed guanine nucleotide exchange. The site, to which suramin analogues bind, overlaps with the docking site for the receptor on the G protein alpha subunit. The structure-activity relationships for inhibition of agonist binding to the A1 adenosine receptor (suramin > NF037 > NF023) and of agonist binding to the inhibition D2 dopamine receptor (suramin = NF037 > NF023 > NF018) differ. Thus, NF037 discriminates between the ternary complexes formed by the agonist-liganded D2 dopamine receptors and those formed by the A1 adenosine receptor with > 10-fold selectivity. Therefore, our results also show that inhibitors can be identified that selectively uncouple specific receptor/G protein tandems.

Thiophosphorylation of the G protein beta subunit in human platelet membranes: evidence against a direct phosphate transfer reaction to G alpha subunits.

A direct phosphate transfer reaction from the G protein beta subunits to either Gs alpha or Gi alpha has been proposed to account for the ability of thiophosphorylated transducin beta gamma-dimers to bidirectionally regulate adenylyl cyclase activity in human platelet membranes. We searched for experimental evidence for this reaction. Incubation of human platelet membranes with [35S]guanosine-5'-(3-O-thio)triphosphate ([35S]GTP gamma S) results in the predominant incorporation of [35S]thiophosphate into a 36-kDa protein, which comigrates with the G protein beta subunit and is immunoprecipitated by a beta subunit-specific antiserum. Thiophosphorylation of the beta subunit is specific for guanine nucleotides and abolished by the histidine-modifying agent diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of [35S]thiophosphorylated beta subunits is accelerated in the presence of GDP, but not ADP, UDP, or guanosine-5'-(2-O-thio)diphosphate. Neither the thiophosphorylation nor the dephosphorylation is sensitive to receptor agonists (alpha 2-adrenergic, A2 adenosine, thrombin, or insulin), and purified G protein alpha subunits do not act as thiophosphate donors. An approach was designed to demonstrate direct thiophosphate transfer to protein-bound nucleotides; platelet membranes were sequentially exposed to NaIO4, NaCNBH3, and NaBH4, an oxidation-reduction step that covalently incorporates prebound nucleotides into proteins. Under these conditions, multiple radiolabeled proteins are visualized on subsequent addition of [35S]GTP gamma S. This reaction is specific because both oxidation and reduction are required and pretreatment of platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyrocarbonate blocks the subsequent labeling in oxidized and reduced membranes. The G protein beta subunit may participate in this thiophosphate transfer reaction. Most important, however, no labeled G protein alpha subunits (Gs alpha and Gi alpha) were recovered by immunoprecipitation from oxidized and reduced membranes subsequent to the addition of [35S]GTP gamma S. Thus, our results clearly rule out the existence of a postulated G protein activation by phosphate transfer reactions, which lead to the formation of GTP from GDP prebound to the alpha subunit.

Species differences in A1 adenosine receptor/G protein coupling: identification of a membrane protein that stabilizes the association of the receptor/G protein complex.

Reconstitution experiments with purified components reproduce the basic characteristics of receptor/G protein coupling, i.e., GTP-sensitive high affinity agonist binding and receptor-promoted GTP binding. However, the interaction of agonists with the A1 adenosine receptor in rat and bovine but not human brain membranes deviates from the ternary complex model since the agonist/receptor/G protein complex cannot be dissociated by high concentrations (> or = 100 microM) of the hydrolysis-resistant analogue GTP gamma S. The reason for this phenomenon referred to as a "tight coupling mode" has remained enigmatic. We show that it is attributable to a distinct membrane protein, which we labeled the coupling cofactor. Extraction of the protein from rat brain membranes with the detergent 3[3-(cholamidopropyl)diamethylammonio]-1-propanamium increased the potency of GTP gamma S by 1000-fold. After extraction, the potency was comparable to that in human brain membrane. Detergent extracts from rat brain membranes were used to resolve the component from solubilized receptors and G protein alpha and beta gamma subunits by sequential DEAE-Sephacel chromatography and Superose gel filtration (molecular weight of approximately 150 kDa in 3[3-(cholamidopropyl)diamethylammonio]-1-propanamium). Coupling cofactor restored guanine nucleotide refractoriness in a concentration-dependent manner to both detergent-extracted rat brain membranes and, albeit with lower affinity, human brain membranes. However, in human brain extracts, cofactor activity was detectable on reconstitution with rat acceptor membranes, indicating an intrinsic difference between rat and human receptors in their ability to interact with the cofactor. With high amounts of coupling cofactor present, GTP gamma S no longer decreased but rather increased agonist affinity. Readdition of partially purified coupling cofactor to acceptor membranes reduced the rate of A1 adenosine receptor-mediated G protein turnover. These observations show that the component identified traps the ternary agonist/receptor/G protein complex in a stable conformation, impedes signaling of the A1 adenosine receptor, and thereby regulates the level of signal amplification.