Distinct interactions of GTP, UTP and CTP with Gs-proteins *

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating Gs-protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support Gs activation. As models, we used S49 wild-type lymphoma cells representing a physiologically relevant system in which the b 2-adrenoceptor ( b 2AR) couples to Gs, and Sf9 insect cell membranes expressing b 2AR-Gs a fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of b 2AR/Gs-coupling than native systems. Nucleoside 5-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP e UTP > CTP > ATP (ineffective). NTPs disrupted high-affinity agonist binding in b 2AR-Gs a in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP e UTP e CTP e GTP. NTPs inhibited b 2AR-Gs a -catalyzed [ g -32P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Gs a than CTP. Collectively, our data indicate that GTP, UTP and CTP differentially interact with Gs-proteins and that transphosphorylation of GDP to GTP is not involved in this G-protein activation. In certain cell systems, intracellular UTP- and CTP concentrations reach ~10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G-protein activation by UTP and CTP can occur physiologically.


Introduction
G-proteins consist of an α-subunit and a βγ-complex and serve as signal transducers between agonist-activated GPCRs 1 and effector systems (1)(2)(3)(4). Upon binding of an agonist, GPCRs undergo a conformational change causing GDP dissociation from G α . GDP dissociation is the rate-limiting step of the G-protein cycle. Agonist-occupied GPCRs then form a ternary complex with the nucleotide-free G-protein. The ternary complex possesses high agonistaffinity. Subsequently, GPCRs promotes binding of GTP to G α . The binding of GTP to G α induces the active conformation of the G-protein, leading to the dissociation of the heterotrimer into G α -GTP and the βγ-complex. Both G α -GTP and βγ can regulate the activity of effector systems. G α possesses GTPase activity. The GTPase cleaves GTP into GDP and P i and, thereby, deactivates the G-protein. G α -GDP and βγ reassociate, completing the G-protein cycle.
Intriguingly, not only the purine nucleotide GTP but also pyrimidine nucleotides exhibit effects on G-proteins. Particularly, various natural and synthetic uracil nucleotides disrupt the complex between the photoexcited light receptor rhodopsin and the retinal G-protein transducin, but the uracil nucleotides are less effective in this regard than the corresponding guanine nucleotides (5). [γ-32 P]GTP hydrolysis-and [ 35 S]GTPγS binding competition studies showed that pyrimidine nucleotides bind to G-proteins with low affinity (5)(6)(7)(8). Moreover, early studies revealed that UTP and CTP support GPCR-mediated AC activation in membranes (9)(10)(11).
However, it remained unclear whether the effects of UTP and CTP on AC were mediated via NDPK, catalyzing the formation of GTP from GDP and NTP (12,13), or via direct interaction of UTP and CTP with G s α (6).
The aim of the present study was to elucidate the mechanism by which UTP and CTP support G s activation. To achieve our aim we have studied AC regulation in S49 membranes.
S49 cells are a widely used and physiologically relevant model system for the analysis of β 2 AR/G s /AC interactions (14)(15)(16)(17). Additionally, we have studied fusion proteins of the β 2 AR with individual G s α isoforms, i.e. β 2 AR-G s αS, β 2 AR-G s αL and β 2 AR-G α olf, expressed in Sf9 insect cells. Fusion proteins provide close proximity of the coupling partners and ensure efficient GPCR/G-protein/effector coupling (18,19). In addition, fusion proteins allow for the analysis of the coupling of a given GPCR to various G α isoforms under defined experimental conditions (20)(21)(22)(23). Here, we report on distinct interactions of GTP, UTP and CTP with G s -proteins.

Experimental Procedures
Materials. The generation of baculoviruses encoding for β 2 AR-G s αS, β 2 AR-G s αL and β 2 AR-G α olf was described elsewhere (20,23,24). 32 P]CTP (~6000 Ci/mmol each) were synthesized as described (5,25). S49 cells were obtained from the Cell Culture Facility of The University of California at San Francisco (San Francisco, CA).
Cell culture and membrane preparation. Sf9 cells were cultured and infected with recombinant baculoviruses as described (20,24,26). S49 cells were grown at 37°C in suspension in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4.5 g/l D-glucose, 2 mM L-glutamine, 1000 U/ml penicillin, 100 µg/ml streptomycin and 10% (vol./vol.) heatinactivated horse serum in a humidified atmosphere containing 7% (vol./vol.) CO 2 . S49 cells were maintained at a density of 0.2-2.0 x 10 6 cells/ml. To inactivate the GTPase of G s α, S49 cells were treated with CTX (1 µg/ml) for 24 h before membrane preparation (27). DMEM medium was from Cellgro Mediatech (Herndon, VA). All other constituents for the culture of S49 cells were obtained from Bio Whittaker (Walkersville, MD). S49-and Sf9 membranes were prepared according to the previously described protocol (24). Membranes were suspended in "binding buffer" (12.5 mM MgCl 2 , 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4) at a concentration of ~1-2 mg of protein/ml and stored at -80°C until use. Immediately prior to [ 3 H]DHA binding-, AC-, NTPase-and NDPK experiments, membrane aliquots were thawed, suspended in binding buffer and centrifuged for 15 min at 4 o C and 15,000 x g to remove, as far as possible, any remaining endogenous nucleotides (23).

Molecular modeling. Potential energy minimization and molecular dynamics simulations
were carried out using the AMBER 6.0 program package (29), using the force field of Cornell et al. (30). Initial models for G s α"Mg 2+ "NTP complexes were based on the coordinates of G s α"Mg 2+ "GTPγS in the complex with the catalytic domains of AC (PDB ID: 1AZS) (31). We replaced the γS substituent of GTPγS by an sp2 hybridized oxygen atom. Models of complexes with UTP and CTP were generated by substitution of the guanine base by uracil and cytosine, respectively, maintaining the glycosyl torsion angle (O4'-C1'-N9-C8) of GTPγS in the starting model. Partial charges assigned to phosphate groups were obtained from Dr. N. Duclert-Savatier (Institut Pasteur, Paris, France) 2 . Protein-bound water molecules observed in the crystal structure were included in the model. The non-bonded cutoff distance was set to 10 Å. The stereochemistry of the Mg 2+ ligand field was restrained as an octahedral complex with the six coordinating oxygen atoms of the βand γ-phosphate oxygens, the hydroxyl groups of Ser 54 (G s αL and G s αS) and Thr 204 (G s αL) and two water molecules, with oxygen-Mg 2+ distances of 2.1 Å. This geometry was maintained by pseudo-van der Waals potentials between all pairs of atoms within the octahedral complex. In constructing models of the G s α"Mg 2+ "UTP-and G s α"Mg 2+ "CTP complexes, we placed a water molecule at the site occupied by the guanine exocyclic C(2) amine in the GTP complex. The included water molecule bridges the uracil exocyclic C (2) keto with the Ο1γ carboxylate oxygen of Asp 295 (G s αL) and Asp 280 (G s αS). The water molecule placed at this site did not move after energy minimization of the model complexes.
Energy relaxations were carried out using the SANDER module of AMBER, using steepest decent minimization for the first 250 cycles, followed by 250 cycles of conjugate gradient minimization. In all cases the total computed potential energy typically declined smoothly from values of -14500 to -16750 kcal•mol -1 , attaining a constant value after 400-450 cycles of minimization. For molecular dynamics simulations, a box of TIP3P water molecules was used to solvate the protein, leaving a 10 Å border between the edge of the box and the closest atoms of the protein. The system was heated to 300 K using the temperature scaling scheme of Berendsen et al. (32) and periodic boundary conditions. Simulations were carried out for 10 ps in steps of 1 fs.

Miscellaneous.
Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Data shown in Figs. 1-5 and Table I were analyzed by non-linear regression, using the Prism III program (GraphPad, Prism, San Diego, CA).

UTP, CTP and GTP differentially support AC activation in S49 membranes. S49 cells
express the β 2 AR, the G s α splice variants G s αS and G s αL and AC (17,33,34). In the absence of added UTP, CTP or GTP, the full βAR agonist ISO at a maximally stimulatory concentration (10 µM) had no stimulatory effect on AC activity in S49 membranes (Fig. 1). However, these experimental conditions do not imply the complete absence of NTP, since the AC assay becomes an efficient AC activator even in the absence of GPCR agonist (1,27,35). In fact, in membranes from CTX-treated S49 cells, GTP was far more efficient at activating AC than were GTP plus ISO in control membranes (Fig. 1C). However, CTX did not increase the potency of GTP (EC 50 , 560 nM; 95% c. i., 310-1000 nM). As was the case with GTP, CTX greatly increased the efficacy of UTP at activating AC (Fig. 1A). The efficacy of UTP at activating AC in membranes from CTX-treated S49 cells amounted to ~70% of the efficacy of GTP. In contrast to the data obtained for GTP, CTX increased the potency of UTP ~4-fold (EC 50 , 21 µM; 95% c. i., 11-38 µM). Although CTX also substantially enhanced AC activation by CTP, this NTP was, nonetheless, much less efficient than UTP and GTP (Fig. 1B). Thus, CTX greatly amplifies the maximum effects of NTPs on basal AC activity without altering their relative efficacies.
Rationale for conducting further studies with Sf9 membranes expressing β 2 AR-G s α fusion proteins. We wished to answer the question whether UTP and CTP, like GTP, disrupt the ternary complex consisting of agonist-occupied β 2 AR and nucleotide-free G s α, whether UTP and CTP inhibit [γ-32 P]GTP hydrolysis by G s α and whether G s α hydrolyzes [γ-32 P]UTP and [γ-32 P]CTP. However, the extent of ternary complex formation in S49 membranes is rather limited (17). Thus, we were concerned that this system would not be sensitive enough to dissect potentially small differences in efficacies of NTPs on high-affinity agonist binding. In addition, S49 membranes are not sensitive models for the analysis of the GTPase activity of G s (36). We were also interested to answer the question whether the different G s α isoforms, i.e. G s αS, G s αL and G α olf, respond similarly to UTP and CTP. However, S49 cells express a mixture of G s αS and G s αL, and the sensitivity of the G s α-deficient S49 cyccells as reconstitution system for the planned studies is limited, too (33,34).
Sf9 membranes expressing β 2 AR-G s α fusion proteins possess a sufficiently high sensitivity for dissecting differential effects of NTPs on ternary complex formation, surpassing the sensitivity of native and recombinant non-fused systems (17,24,26). Additionally, β 2 AR-G s α fusion proteins are sensitive models for NTPase studies (24,26). Moreover, β 2 AR-G s α fusion proteins are suitable systems for dissecting biochemical differences between G s α isoforms (20,22,23). Based on these considerations, we decided to conduct all further studies with β 2 AR-G s α fusion proteins. β 2 AR-G α olf (EC 50 , 19 µM; 95% c. i., 7-62 µM). The efficacy of CTP at disrupting the ternary complex amounted to less than 25% of the efficacy of GTP, preventing us from calculating meaningful EC 50 values for CTP. Of particular importance was the finding that ATP did not disrupt the ternary complex in membranes expressing β 2 AR-G s αS or β 2 AR-G α olf.
To corroborate the conclusion that the order of efficacy of NTPs at disrupting the ternary complex is GTP > UTP > CTP, we determined the extent of ternary complex formation in β 2 AR-G s αL by competing [ 3 H]DHA binding by the full agonist ISO in the absence and presence of NTPs at a saturating concentration (1 mM). Fig. 3 shows the agonist-competition curves and Table I summarizes the non-linear regression analysis of these binding experiments. As reported before (20,24), ISO inhibited [ 3 H]DHA binding according to a biphasic function, with ~35% of the β 2 ARs being in a state of high agonist-affinity (Fig. 3A). GTP (1 mM) substantially shifted the ISO-competition curve to the right and converted the competition curve into a steep monophasic function, reflecting complete disruption of the ternary complex. In contrast, in the presence of 1 mM UTP, the agonist-competition curve was still biphasic (~10% high-affinity agonist binding remaining), and the low-affinity component of the competition curve was not shifted as far to the right as with GTP (Fig. 3B). These data confirm the notion that UTP disrupted the ternary complex only incompletely. CTP had only minimal inhibitory effects on ternary complex formation in membranes expressing, β 2 AR-G s αL i.e. the fraction of highaffinity binding sites was not decreased compared to control conditions ( Fig. 3C and Table I). In addition, CTP increased the K i values for ISO only slightly relative to control. Taken together, the analysis of the effects of NTPs on ternary complex formation in membranes expressing β 2 AR-G s α fusion proteins clearly showed that the order of efficacy is GTP > UTP > CTP > ATP (ineffective).

Competition of β 2 AR-G s α-catalyzed [γ-32 P]GTP hydrolysis by GTP, UTP and CTP.
In order to address the question whether UTP and CTP bind to the nucleotide-binding pocket of G s α, we stimulated G s α-catalyzed [γ-32 P]GTP hydrolysis in β 2 AR-G s α fusion proteins by ISO and competed [γ-32 P]GTP hydrolysis with unlabeled NTPs. For all three β 2 AR-G s α fusion proteins we obtained monophasic competition curves, indicating that [γ-32 P]GTP and NTPs competed for binding to a single site (Fig. 4). The We observed certain differences in the effects of UTP and CTP at the various fusion proteins in the AC assay. Specifically, the maximum agonist-stimulated AC activities achieved with CTP in membranes expressing β 2 AR-G s αS and β 2 AR-G s αL amounted to ~65% of that obtained with UTP, whereas in membranes expressing β 2 AR-G α olf, the maximum AC activity achieved with CTP amounted to only ~40% of the activity obtained with UTP (compare Figs. 5A with 5B, Figs. 5D with 5E, and Figs. 5G with 5H, respectively). There were also differences in the relative stimulatory effects of ISO in the individual fusion proteins in the presence of different NTPs. In membranes expressing β 2 AR-G s αS, the maximum stimulatory effects of ISO amounted to 74% (UTP), 55% (CTP) and 57% (GTP), respectively. The corresponding values for β 2 AR-G s αL were 74% (UTP), 115% (CTP) and 60% (GTP), respectively. For β 2 AR-G α olf the stimulations were 195% (UTP), 77% (CTP) and 206% (GTP), respectively.
While GTP exhibited similar potency (EC 50 ~1 µM) and relative efficacy as NDPK substrate in

Molecular modeling of the interactions of GTP, UTP and CTP with G s α.
In order to provide an explanation for the different affinities and efficacies of NTPs at G s -proteins we constructed models for the GTP-, CTP-and UTP complexes of G s α based on the structure of the G s α"Mg 2+ "GTPγS complex (31) and subjected the complexes to potential energy minimization. However, none of the energy-minimized G s α"Mg 2+ "NTP models differed substantially from the corresponding G s α"Mg 2+ "GTPγS complex (0.15Å -0.18Å root mean square deviation between pairs of corresponding Cα atoms) (data not shown). Furthermore, the position, orientation and conformation of NTP"Mg 2+ were unaltered after energy minimization. Hence, energy minimization experiments did not provide an explanation for the differences in affinity and efficacy of NTPs at G s α.
We then decided to investigate the molecular models of the GTP-, UTP-and CTP complexes of G s α by molecular dynamics simulation, After 10 ps simulation at 300 K, the G s α"Mg 2+ "GTP-, G s α"Mg 2+ "UTP-and G s α"Mg 2+ "CTP models diverged from the corresponding G s α"Mg 2+ "GTPγS complex by 0.94 Å, 0.95 Å, and 1.15 Å, root mean square, respectively, over all Cα pairs. The deviations were smaller (0.6-0.8 Å) for the set limited to Cα atoms within 6 Å of the NTPs. The models did not change further after 500 cycles of energy minimization. All of the guanine ring-G s α hydrogen bonds and van der Waals interactions observed in the structure of the G s α"Mg 2+ "GTPγS complex (31, 37) were retained in the model of the G s α"Mg 2+ •GTP complex after molecular dynamics simulation (Fig. 7A), although the ribosyl group shifted ~1 Å.
Likewise, the molecular geometry of the triphosphate moiety and its interaction with Mg 2+ were unperturbed, indicating that the simulation with GTP preserves experimentally observed properties of the interaction of G s α with GTPγS.
In contrast, the molecular dynamics simulations of the G s α"Mg 2+ "UTP-and G s α"Mg 2+ "CTP complexes had different outcomes. The position and orientation of the uracil ring of UTP remained near the starting position after 10 ps of dynamics simulation (Fig. 7B). The N3 imine stayed in position to donate a hydrogen bond to the Oδ2-carboxylate oxygen of Asp 295, and the hydrogen bond network involving the 2-keto oxygen, the imposed water molecule and Oδ1 of Asp 295 were intact, although with sub-optimal geometry. In contrast, the cytosine ring of CTP rotated ~10° from its initial orientation (Fig. 7C) Table I) and support AC activation by unmodified and ADP-ribosylated G s α (Figs. 1   and 5). An explanation for those observations could be transphosphorylation of endogenous GDP to GTP by UTP or CTP via NDPK (12,13,38), but several findings argue against this notion.
First, the concentration of G s αS-GDP in washed Sf9 membranes is higher than the concentration of G s αL-GDP and G α olf-GDP (20,23). Thus, for transphosphorylation, membranes expressing β 2 AR-G s αS should have been a considerably more efficient system than membranes expressing β 2 AR-G s αL and β 2 AR-G α olf. However, UTP and CTP were not more efficient at disrupting the ternary complex and supporting AC activation in membranes expressing β 2 AR-G s αS than in membranes expressing β 2 AR-G s αL and β 2 AR-G α olf (Figs. 2 and 5). Second, a given NTP should have had the same relative efficacies in the AC-and highaffinity agonist binding assays. However, UTP was less efficient than GTP at disrupting the ternary complex in β 2 AR-G s α fusion proteins (Figs. 2 and 3 and Table I). In contrast, UTP was equally efficient as GTP at supporting ISO-stimulation of AC in Sf9 membranes expressing β 2 AR-G s α fusion proteins (Fig. 5). Third, in S49-and Sf9 membranes, ATP was a potent (EC 50 ~5 µM) and efficient phosphoryl group donor for GTP formation (Fig. 6), but we failed to detect a stimulatory effect of ISO on AC activity in S49-and Sf9 membranes in the presence of ATP (40 µM) (Figs. 1 and 5). Fourth, CTP and UTP were similarly efficient NDPK substrates in S49 membranes (Fig. 6A), but CTP was much less efficient than UTP at supporting AC activation in this system (Figs. 1A and 1B). Finally, in S49-and Sf9 membranes UTP was a more efficient phosphoryl group donor for [ 3 H]GTP formation than GTP (Fig. 6), but UTP was not more efficient than GTP with respect to disruption of the ternary complex and AC activation.
Evidence that GTP, UTP and CTP stabilize distinct conformations of G s α. We then considered the hypothesis that UTP and CTP exert their effects on AC and ternary complex formation directly by binding to the nucleotide-binding pocket of G s α. Indeed, UTP and CTP have already been shown to bind with low affinity to various G-proteins including G s (5)(6)(7)(8). In agreement with the results of the earlier studies, our GTPase competition studies with β 2 AR-G s α fusion proteins showed that NTPs bind to G s αS, G s αL and G α olf in the order of affinity GTP > UTP > CTP (Fig. 4). We noted that the apparent affinities of UTP and CTP in the agonist binding-and AC studies with fusion proteins (Figs. 2 and 5) were considerably higher than in the GTPase competition studies (Fig. 4). An important difference between these experiments is that the GTPase studies were conducted in the presence of GTP, whereas the agonist bindingand AC studies were conducted in the absence of GTP (see Experimental Procedures). These data raise the intriguing hypothesis that in the presence of GTP, access of UTP and CTP to G s α is restricted. Evidence for restricted access of nucleotides to G-proteins was already obtained in a previous study (39).
If the NTPs studied had stabilized the same conformation in G s α we would have expected NTPs to exhibit the same maximum effects on ternary complex formation and AC activation at saturating concentrations. However, this was clearly not the case. The overall order of efficacy of NTPs with respect to these parameters was GTP e UTP > CTP (Figs. 1-3 and 5).
The enhancing effects of CTX on maximum AC activation by NTPs (Fig. 1) are particularly intriguing. CTX unmasks a strong stimulatory effect of GTP on AC by blocking the GTPase activity of G s α (1,27,35). Our present data are in agreement with this concept (Fig. 1C). CTX also greatly enhanced the stimulatory effects of UTP and CTP on AC activity (Figs. 1A and 1B), suggesting that G s α hydrolyzes UTP and CTP as well. However, we could not obtain evidence for the presence of UTPase-and CTPase activity in β 2 AR-G s α-proteins although Sf9 membranes expressing these proteins are highly sensitive systems to detect NTPase activity (24,26). These data imply that the mechanism of deactivation of G s α-UTP and G s α-CTP is NTP dissociation rather than NTP hydrolysis. Our data indicate that CTX-catalyzed ADPribosylation induces a conformational change in G s α that is independent of the well-established GTPase inhibition and allows UTP and CTP to interact more productively with the G-protein. In support of this hypothesis is the fact that CTX-catalyzed ADP-ribosylation of G s α increased the apparent affinity of the G-protein for UTP (Fig. 1A). In contrast to UTP, we did not observe an increase of affinity of G s α for GTP following CTX-catalyzed ADP-ribosylation (Fig. 1C).
Taken together, our data indicate that GTP, UTP and CTP interact with G s -proteins in nonidentical fashions.
The molecular dynamics simulations are consistent our experimental data. Specifically, the simulation experiments indicate that CTP binds with lower affinity to G s α than UTP because optimal interactions require a conformational change in the protein with no net gain in stabilizing interactions relative to UTP (Fig. 7). In fact, CTP binds to G s -proteins with lower affinity than UTP (Fig. 4). In addition, UTP and CTP are expected to bind to G s α with lower affinity than GTP because they form fewer stabilizing hydrogen bonds to the G-protein. In particular, hydrogen bond analogous to that between the guanine N(7) and Asn 292 (31, 37, 40) cannot be formed with uracil and cytosine. The experimental data are in agreement with the modeling studies (Figs. 1, 2, 4 and 5).
Compared to CTP, UTP is more easily accommodated within the binding site of G s α and forms more hydrogen bonds with the protein, assuming the participation of a water molecule captured from the solvent. The differences in interactions of GTP, UTP and CTP with G s α could be interpreted in such a way that each of the NTPs stabilizes a distinct conformation of G s α or that certain NTPs stabilize less productive states of the G-protein than GTP. Specifically, G s α-GTP possesses a conformation that is highly efficient at disrupting the high-affinity interaction with the agonist-occupied β 2 AR and at activating AC, whereas G s α-CTP possesses a conformation that is inefficient in these regards. Of particular interest, G s α-UTP possesses a conformation that is similarly efficient as G s α-GTP at activating AC (Fig. 5) but less efficient than G s α-GTP at disrupting the high-affinity interaction with the agonist-occupied β 2 AR (Figs. 2 and 3 and Table I). In agreement with our present data on G s , guanine nucleotides are more efficient at disrupting the complex between photoexcited rhodopsin and transducin than the corresponding uracil nucleotides (5). These data indicate that G-proteins do not simply act as on/off switches but rather exist in states with different functional capacities that are differentially stabilized by NTPs.
Although the overall pattern of effects of GTP, UTP and CTP at the various β 2 AR-G s α fusion proteins was similar (Figs. 2 and 5), we noted that CTP was particularly effective at supporting ISO-stimulation of AC in Sf9 membranes expressing β 2 AR-G s αL (Fig. 5E). These data indicate that there are subtle differences in the conformations of G s αS-CTP, G s αL-CTP and G α olf-CTP, exhibiting different efficacies at activating AC relative to the corresponding conformations of G s α-GTP and G s α-UTP. Studies with hydrolysis-resistant phosphorothioate analogs of UTP and CTP will be important to substantiate the concept of distinct active G s α conformations.
Physiological and pathological relevance of G-protein activation by pyrimidine nucleotides. Differential G s -activation by NTPs was observed in Sf9 membranes expressing β 2 AR-G s α fusion proteins (Figs. 2, 3 and 5) and in a physiological system, the S49 membranes. The effects of UTP and CTP on fused and non-fused G s -proteins were observed at concentrations e 10 µM (Figs. 1-5). The bulk intracellular UTP-and CTP concentrations in various neuronal and astroglial cells may be as high as ~10 nmol/mg of protein and may exceed the intracellular GTP concentrations by up to ~two-fold (41)(42)(43)(44). Thus, it is likely that at least in some cellular systems the intracellular UTP-and CTP concentrations are sufficiently high for allowing these NTPs to activate G-proteins under physiological conditions, provided that the access of UTP and CTP to G α is not severely restricted.
CTX-catalyzed ADP-ribosylation substantially increases both the potency and efficacy of UTP at activating AC (Fig. 1A). This effect of CTX appears to be due to a conformational change in G s α rather than due to GTPase/NTPase inhibition. In other words, CTX-catalyzed ADP-ribosylation may optimize the conformation of G s α to bind UTP. Thus, enhanced G s activation by UTP may contribute to the clinical symptoms of cholera.
Lesch-Nyhan syndrome is caused by a defect of hypoxanthine-guanine phosphoribosyltransferase, a key enzyme in the purine salvage pathway (45). This enzyme defect results in a decrease in intracellular GTP concentration and increases in intracellular UTP-and CTP concentrations (43,44,(46)(47)(48). Intriguingly, patients with Lesch-Nyhan syndrome have alterations in the regulation of multiple neurotransmitter systems including the adrenergic system (49)(50)(51)(52)(53)(54) and severe neuropsychiatric abnormalities (45). Given the fact that Lesch-Nyhan syndrome is associated with changes in intracellular GTP-, UTP-and CTP concentrations and that these NTPs differ in their effects on G-protein-mediated signaling, one could envisage that GPCR/G-protein/effector coupling is altered in Lesch-Nyhan syndrome, resulting in impaired development and function of the central nervous system.         represents the included water molecule. In panels B and C, the G s α"Mg 2+ "UTP-and G s α"Mg 2+ "CTP complexes, respectively, are superimposed on the G s α"Mg 2+ "GTP complex that is shown as charcoal stick model. Table I