GDP Affinity and Order State of the Catalytic Site Are Critical for Function of Xanthine Nucleotide-selective Galpha s Proteins*

Andreas GilleDagger , Katharina Wenzel-SeifertDagger , Michael B. Doughty§, and Roland SeifertDagger ||

From the Departments of Dagger  Pharmacology and Toxicology and § Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66045-7582 and  Department of Chemistry and Physics, Southeastern Louisiana University, Hammond, Louisiana 70402-0878

Received for publication, October 3, 2002, and in revised form, December 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Xanthine nucleotide-selective small GTP-binding proteins with an Asp/Asn mutation are valuable for the analysis of individual GTP-binding proteins in complex systems. Similar applications can be devised for heterotrimeric G-proteins. However, Asp/Asn mutants of Galpha o, Galpha 11, and Galpha 16 were inactive. An additional Gln/Leu mutation in the catalytic site, reducing GTPase activity and increasing GDP affinity, was required to generate xanthine nucleotide-selective unspecified G-protein alpha -subunit (Galpha ). Our study aim was to generate xanthine nucleotide-selective mutants of Galpha s, the stimulatory G-protein of adenylyl cyclase. The short splice variant of Galpha s (Galpha sS) possesses higher GDP affinity than the long splice variant (Galpha sL). Nucleoside 5'-[gamma -thio]triphosphates (NTPgamma Ss) and nucleoside 5'-[beta ,gamma -imido]triphosphates effectively activated a Galpha sS mutant with a D280N exchange (Galpha sS-N280), whereas nucleotides activated a Galpha sL mutant with a D295N exchange (Galpha sL-N295) only weakly. The Gln/Leu mutation enhanced Galpha sL-N295 activity. NTPgamma Ss activated Galpha sS-N280 and a Galpha sL mutant with a Q227L and D295N exchange (Galpha sL-L227/N295) with similar potencies, whereas xanthosine 5'-triphosphate and xanthosine 5'-[beta ,gamma -imido]triphosphate were more potent than GTP and guanosine 5'-[beta ,gamma -imido]triphosphate, respectively. Galpha sS-N280 interacted with the beta 2-adrenoreceptor and exhibited high-affinity XTPase activity. Collectively, (i) Galpha sS-N280 is the first functional xanthine nucleotide-selective Galpha with the Asp/Asn mutation alone; (ii) sufficiently high GDP affinity is crucial for Galpha Asp/Asn mutant function; (iii) with nucleoside 5'-triphosphates and nucleoside 5'-[beta ,gamma -imido]triphosphates, Galpha s-N280 and Galpha sL-L227/N295 exhibit xanthine nucleotide selectivity, whereas NTPgamma Ss sterically perturb the catalytic site of Galpha and annihilate xanthine selectivity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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G-proteins are heterotrimeric (alpha beta gamma -structure) and mediate transmembrane signal transfer between receptors and effectors (1-3). Activated receptor promotes GDP dissociation from Galpha .1 GDP dissociation is the rate-limiting step of the G-protein cycle. Subsequently, receptor catalyzes GTP binding to Galpha . GTP binding to Galpha induces the active conformation of the G-protein, leading to the dissociation of the heterotrimer into Galpha -GTP and the beta gamma -complex. Both Galpha -GTP and beta gamma regulate the activity of effector systems. Galpha possesses GTPase activity. The GTPase hydrolyzes GTP into GDP and Pi and thereby deactivates the G-protein. Galpha -GDP and beta gamma reassociate, completing the G-protein cycle. The GTP analogs GTPgamma S and GppNHp are GTPase-resistant and persistently activate G-proteins (1, 4). The gamma -thiophosphate of GTPgamma S is bulkier than the gamma -phosphate of GppNHp/GTP (4, 5). As a result of these chemical differences, GTPgamma S, unlike GppNHp, sterically perturbs the structure of the catalytic site of Galpha (6). These crystallographic data imply that Galpha -GppNHp resembles Galpha -GTP more closely than Galpha -GTPgamma S.

Galpha consists of the ras-like domain that is structurally similar to small GTP-binding proteins and the alpha -helical domain that is unique in Galpha . The two domains embed the nucleotide-binding pocket (3, 7). Nucleotide binding to Galpha involves several hydrogen and ionic bonds. Of particular importance for guanine selectivity is an aspartate that is conserved among small GTP-binding proteins and Galpha (3, 7-11). Exchange of this aspartate against asparagine (Asp/Asn mutation) in small GTP-binding proteins switches base selectivity from guanine to xanthine. Such mutants are valuable to study a specific small GTP-binding protein in complex systems containing multiple GTP-binding proteins (8-11). Similar applications can be devised for G-proteins. Unexpectedly, Asp/Asn mutants of Galpha o, Galpha 11, and Galpha 16 were inactive (5, 12, 13). However, the additional exchange of a conserved glutamine against leucine (Gln/Leu mutation) in the catalytic site of Galpha (3, 7) resulted in active Galpha with the expected xanthine nucleotide selectivity (5, 12, 13). The Gln/Leu mutation reduces GTPase activity and increases GDP-affinity of Galpha (14, 15).

Our study aim was to generate xanthine nucleotide-selective mutants of Galpha s. Galpha s mediates coupling of the beta 2AR to AC (1, 2). Galpha s exists as two splice variants, Galpha sS and Galpha sL, with Galpha sS possessing ~2-3-fold higher GDP-affinity than Galpha sL (16). We generated Galpha sS-N280, Galpha sS-L212, Galpha sS-L212/N280, Galpha sL-N295, Galpha sL-L227, and Galpha sL-L227/N295 and analyzed these Galpha s mutants with guanine-, hypoxanthine-, and xanthine-substituted NTPs, NTPgamma Ss, and NppNHps. We used Sf9 cells as an expression system because this system is suitable for Galpha s analysis, and there is no interference of nucleoside diphosphokinase-mediated trans(thio)phosphorylation reactions with the effects of NTPs/NTPgamma Ss on Galpha s (17-19).

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Materials-- Baculovirus encoding Galpha sS was kindly donated by Dr. A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). Baculovirus encoding Galpha sL was kindly donated by Dr. H. Bourne (Department of Pharmacology, University of California, San Francisco, CA). [gamma -32P]GTP (6000 Ci/mmol), [alpha -32P]ATP (3000 Ci/mmol), [32P]Pi (8500-9000 Ci/mmol), and [3H]dihydroalprenolol (85-90 Ci/mmol) were from PerkinElmer Life Sciences. Unlabeled ATP (special quality, <0.01% (w/w) GTP as assessed by high pressure liquid chromatography, # 519 979), GDP, GTP, GTPgamma S, GppNHp, adenosine 5'-[beta ,gamma -imido]triphosphate, and adenosine 5'-[gamma -thio]triphosphate were of the highest quality available and were obtained from Roche Molecular Biochemicals. [gamma -32P]XTP (~6000 Ci/mmol) was synthesized as described previously (20). ISO, XDP, IDP, bovine liver nucleoside diphosphokinase, and the M1 monoclonal antibody (anti-FLAG Ig) were from Sigma. IppNHp and XppNHp were obtained from JenaBioscience (Jena, Germany). ICI 118,551 was from RBI (Natick, MA). Pfu DNA polymerase was from Stratagene (La Jolla, CA). All restriction enzymes and DNA-modifying enzymes were from New England Biolabs (Beverly, MA). The anti-Galpha s Ig (C-terminal) was from Calbiochem (La Jolla, CA).

NTPgamma S Synthesis-- ITPgamma S and XTPgamma S were synthesized by nucleoside diphosphokinase-catalyzed transthiophosphorylation as described previously (21), with modifications. Briefly, reaction mixtures contained 10 mM IDP or XDP and 5 mM adenosine 5'-[gamma -thio]triphosphate in 30 mM Tris/HCl, pH 8.0, supplemented with 5.0 mM dithiothreitol and 5.0 mM MgCl2 in a total volume of 1.0 ml. The reaction was initiated by the addition of 78 IU nucleoside diphosphokinase and conducted at 37 °C for 24-48 h until the reaction equilibrium resulted in maximum NTPgamma S product. The product NTPgamma S was then purified by fast protein liquid chromatography mono Q ion-exchange chromatography using either a 30 min linear gradient from 0.1-2 M ammonium acetate, pH >8.0, or by isocratic elution in the same buffer (the gradient method allowed the recovery of starting IDP/XDP). The collected product peak was lyophilized and, if necessary, repurified using a shallow gradient. The final product was analyzed for purity by both isocratic and gradient elution from the same chromatography systems used for the purification. The pH > 8.0 ammonium acetate purification buffer produced synthetic NTPgamma S of >98% purity based on fast protein liquid chromatography elution. At lower pH, the product was a mixture of NTPgamma S and IDP/XDP at a ratio of >88:12. Purified products contained 0.75-1.2 µmol of NTPgamma S as assessed by UV absorption at lambda max relative to IDP/XDP standard.

Construction of cDNAs for Galpha s Mutants-- cDNAs for Galpha s mutants were generated by overlap extension PCR (22, 23) using pGEM-3Z-beta 2AR-Galpha sL as template. Sense and antisense primers encoding for the Q227L mutation (L227) created a diagnostic BanII site; sense and antisense primers encoding for the D295N mutation (N295) resulted in the loss of a BglII site. For generation of Galpha sL-L227/N295 cDNA, pGEM-beta 2AR-Galpha sL-L227 served as template, using the primers for generation of the D295N mutation. Recombinant pGEM-3Z-beta 2AR-Galpha sL plasmids were digested with EcoRI and XbaI and cloned into pVL1392-beta 2AR-Galpha sL digested with EcoRI and XbaI. To generate the corresponding Galpha sS cDNAs, the NheI/EcoRI fragment of pGEM-3Z-beta 2AR-Galpha sS replaced the NheI/EcoRI fragment of pGEM-3Z-beta 2AR-Galpha sL mutants. For generation of pVL1392-Galpha s plasmids devoid of beta 2AR cDNA, pVL1392-beta 2AR-Galpha s plasmids were digested with SacI and PflMI to eliminate beta 2AR cDNA. The noncoherent DNA ends were filled with Klenow fragment, and the pVL1392-Galpha s plasmids were religated. Extensive restriction enzyme diagnostics and enzymatic sequencing confirmed all mutations.

Sf9 Cell Culture and Membrane Preparation-- Sf9 cells were cultured in 250-ml disposable Erlenmeyer flasks at 28 °C under shaking at 125 rpm in SF 900 II medium (Invitrogen). Recombinant baculoviruses were generated in Sf9 cells using the BaculoGOLD transfection kit (Pharmingen) as described previously (23). For protein expression, Sf9 cells were infected with 1:100 dilutions of high titer baculovirus stocks and cultured for 48 h (17). Sf9 membranes were prepared as described previously (17). Membranes were suspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4) at a concentration of ~1-2 mg protein/ml and stored at -80 °C until use. Expression levels of beta 2AR-Galpha s fusion proteins were determined by [3H]dihydroalprenolol saturation binding (17). Immediately before AC and NTPase experiments, membrane aliquots were thawed and centrifuged for 15 min at 4 °C and 15,000 × g to remove, as far as possible, endogenous nucleotides (23).

AC Activity-- The determination of AC activity in Sf9 membranes was performed as described previously (23). Briefly, tubes (30 µl) contained membranes (20-40 µg protein/tube), 5 mM MgCl2, 0.4 mM EDTA, 30 mM Tris/HCl, pH 7.4, and nucleotides at various concentrations. Some experiments were conducted in the presence of ISO or ICI 118,551. Tubes were incubated for 3 min at 37 °C before the addition of 20 µl of reaction mixture containing (final) [alpha -32P]ATP (0.5-1.5 µCi/tube) plus 40 µM ATP, 2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU pyruvate kinase, 1.0 IU myokinase, and 0.1 mM cAMP. Reactions were conducted for 20 min. Stopping of reactions and separation of [alpha -32P]ATP from [32P]cAMP were performed as described previously (23). In some experiments, the incubation temperature was varied from 16-37 °C.

NTPase Activity-- High-affinity GTPase activity in Sf9 membranes expressing beta 2AR-Galpha s fusion proteins was determined as described previously (23). Briefly, tubes (80 µl) contained membranes (10 µg protein/tube), 1.0 mM MgCl2, 0.1 mM EDTA, 0.1 mM ATP, 1 mM adenosine 5'-[beta ,gamma -imido]triphosphate, 5 mM creatine phosphate, 40 µg creatine kinase, 30 nM to 10 µM unlabeled GTP, 10 µM ISO, and 0.05% (w/v) bovine serum albumin in 50 mM Tris/HCl, pH 7.4. Reaction mixtures were incubated for 3 min at 25 °C before the addition of 20 µl of [gamma -32P]GTP (2.0 µCi/tube). Nonenzymatic [gamma -32P]GTP hydrolysis was determined in the presence of a large excess of unlabeled GTP (1 mM) and amounted to <1% of total [gamma -32P]GTP hydrolysis. Reactions were conducted for 20 min. Stopping of reactions and recovery of [32P]Pi were performed as described previously (23). XTPase activity was determined as GTPase activity, except that XTP and [gamma -32P]XTP were used.

Immunoblot Analysis-- Membrane proteins were separated on SDS-polyacrylamide gels containing 10% (w/v) acrylamide. Proteins were transferred onto Immobilon-P transfer membranes (Millipore, Bedford, MA). Membranes were reacted with anti-Galpha s Ig or anti-FLAG Ig (1:1000 each). Immunoreactive bands were visualized by donkey anti-rabbit IgG (anti-Galpha s Ig) or sheep anti-mouse IgG (anti-FLAG Ig) coupled to peroxidase at 1:1000 dilutions, using o-dianisidine and H2O2 as substrates. Immunoblots were scanned using a Molecular Imager FX and evaluated with the Quantity One 4.3 software (Bio-Rad, Hercules, CA), using beta 2AR-Galpha s fusion proteins (25-75 µg/lane) expressed at defined levels ([3H]dihydroalprenolol saturation binding) as standard.

Miscellaneous-- Protein concentrations were determined with the Bio-Rad DC protein assay kit (Bio-Rad). Data shown in Figs. 2-4 were analyzed by nonlinear regression using the Prism 3.02 software (GraphPad, Prism, San Diego, CA).

    RESULTS
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ABSTRACT
INTRODUCTION
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Expression of Galpha s- and beta 2AR-Galpha s Proteins in Sf9 Membranes-- In mammalian cells, Galpha sS and Galpha sL possess apparent molecular masses of 45 and 52 kDa, respectively (24-26). The same was true for Galpha sS and Galpha sL expressed in Sf9 membranes (Fig. 1A). In agreement with a recent report (27), Galpha sL expressed in Sf9 membranes also showed a ~40-kDa proteolytic fragment. In contrast, Galpha sS did not exhibit major proteolytic fragments. However, in membranes expressing Galpha sS-L212, Galpha sS-N280, and Galpha sS-L212/N280, a prominent ~39-kDa proteolytic fragment became apparent (Fig. 1B). In membranes expressing the corresponding Galpha sL mutants, the ~40-kDa fragment was present, and an additional ~46-kDa proteolytic fragment emerged. These data indicate that Galpha s and Galpha s mutants possess different conformations, i.e. proteases access Galpha s mutants more readily than Galpha s. Galpha s and Galpha s mutants were expressed at similar levels (9-15 pmol/mg). We also expressed beta 2AR-Galpha s fusion proteins. Fusion proteins allow for the sensitive analysis of beta 2AR/Galpha s coupling, particularly with respect to NTP hydrolysis (17, 18). beta 2AR-Galpha sS and beta 2AR-Galpha sL proteins exhibited the expected molecular masses of ~100 and 106 kDa, respectively (Fig. 1C) (22). The expression levels of beta 2AR-Galpha s proteins ranged between 4 and 7 pmol/mg. Collectively, Galpha s and Galpha s mutants in the nonfused and fused state were expressed at similar levels in Sf9 membranes. For brevity, we only show the AC data with nonfused Galpha s. Studies with beta 2AR-Galpha s proteins yielded essentially the same results.


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Fig. 1.   Analysis of the expression of Galpha s- and beta 2AR-Galpha s proteins in Sf9 membranes. Sf9 cells were infected with the baculoviruses indicated below A-C and incubated for 48 h before membrane preparation. Sf9 cell membranes (60 µg protein/lane) were separated on SDS gels containing 10% (w/v) acrylamide as described under "Experimental Procedures." Proteins were transferred onto Immobilon-P transfer membranes and probed with anti-Galpha s Ig (C-terminal) (A and B) or anti-FLAG Ig (C). The expression levels of nonfused Galpha s proteins were estimated using beta 2AR-Gsalpha L (7.0 pmol/mg as assessed by [3H]dihydroalprenolol saturation binding) as standard. Numbers on the left indicate molecular masses (in kDa) of marker proteins.

Regulation of AC Activity by NTPs, NTPgamma Ss, and NppNHps in Sf9 Membranes Expressing Galpha sL and Galpha sL Mutants-- GTP, ITP, and XTP were essentially devoid of stimulatory effects on AC in membranes expressing Galpha sL, Galpha sL-N295, and Galpha sL-L227 (Fig. 2, A, B, and D). A striking difference among those three constructs was the extremely high basal AC activity in the absence of GTP, ITP, or XTP in membranes expressing Galpha sL-L227. This finding could be explained by the GTPase deficiency brought about by the Gln/Leu mutation (14, 15). However, the Gln/Leu mutation also increases the GDP affinity of Galpha sL (14, 15), and Galpha s-GDP activates AC as efficiently as Galpha s-GTPgamma S, provided that the concentration of Galpha s-GDP is sufficiently high (28). As will be shown at the end of "Results," the beta 2AR agonist ISO efficiently reduces AC activity in the absence of GTP in membranes co-expressing the beta 2AR plus Galpha sL-L227/N295 through GDP dissociation and generation of nucleotide-free Galpha s. Nucleotide-free Galpha s is less efficient at activating AC than Galpha s-GDP (22, 23). These data indicate that the increase in GDP affinity induced by the Gln/Leu mutation critically contributes to the high basal AC activity in membranes expressing such mutants.


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Fig. 2.   Effects of NTPs, NTPgamma Ss, and NppNHps on AC activity in Sf9 membranes expressing Galpha sL proteins. AC activity in Sf9 membranes was determined as described under "Experimental Procedures." Reaction mixtures contained Sf9 membranes (20-40 µg protein/tube) expressing Galpha sL (A, E, and I), Galpha sL-N295 (B, F, and J), Galpha sL-L227/N295 (C, G, and K), or Galpha sL-L227 (D, H, and L) in the presence of NTPs (A-D), NTPgamma Ss (E-H), or NppNHps (I-L) at the concentrations indicated on the abscissa. Log -10 designates the absence of added guanine, hypoxanthine, or xanthine nucleotide. Note that because of the high AC activities with membranes expressing Galpha sL-L227, the scale of the y axis in D, H, and L is different from that in the other panels. Data were analyzed by nonlinear regression and best fitted to sigmoid concentration-response curves. Data shown are the means ± S.D. of three to six experiments performed in duplicates.

Galpha sL-N295 and Galpha sL-L227/N295 were expected to possess decreased GDP affinity (5, 12, 13) and basal AC activity relative to Galpha sL and Galpha sL-L227, respectively. The experimental data were in agreement with these assumptions (Fig. 2, A-D). In contrast to membranes expressing Galpha sL and Galpha sL-N295, NTPs significantly increased AC activity in membranes expressing Galpha sL-L227/N295, compatible with a reduction of NTPase activity of Galpha sL-L227/N295 (14, 15). In fact, ISO had virtually no stimulatory effect on GTP and XTP hydrolysis in membranes expressing beta 2AR-Galpha sL-L227/N295 (data not shown). In agreement with data for the rab5-N136 mutant (29), the order of potency of NTPs at Galpha sL-L227/N295 was XTP > GTP > ITP.

In membranes expressing Galpha sL, NTPgamma Ss stimulated AC activity in the expected order of potency, GTPgamma S > ITPgamma S > XTPgamma S (Fig. 2E) (18, 29). In membranes expressing Galpha sL-N295, the order of potency was XTPgamma S > ITPgamma S > GTPgamma S, but the maximum AC activities in those membranes amounted to only ~20% of the AC activities in membranes expressing Galpha sL. These data indicate that Galpha sL-N295, despite its efficient expression in Sf9 membranes (Fig. 1B), exhibited only low functional activity. Routinely, we conducted AC assays at 37 °C. Intriguingly, the Galpha sL-A366S mutant, which, like Galpha sL-N295, possesses lower GDP affinity than Galpha sL, denatures at 37 °C and requires temperatures of <33 °C to be active (30). Therefore, we varied the incubation temperature in the AC assay from 16 °C to 37 °C, but these maneuvers did not increase AC activation by Galpha sL-N295 (data not shown). These data indicate that Galpha sL-N295 was largely expressed as a functionally inactive protein, although the incubation temperature of Sf9 cells was rather low (28 °C). The poor activity of Galpha sL-N295 is reminiscent of the properties of the analogous Asp/Asn mutants of Galpha o, Galpha 11, and Galpha 16 (5, 12, 13). In Galpha o, Galpha 11, and Galpha 16 Asp/Asn mutants, the additional Gln/Leu mutation resulted in XTPgamma S-selective Galpha mutants (5, 12, 13). However, the switch in base selectivity in Galpha sL-L227/N295 was incomplete, i.e. GTPgamma S, ITPgamma S, and XTPgamma S were similarly potent (Fig. 2G). In membranes expressing Galpha sL-L227, NTPgamma Ss were similarly potent at moderately reducing AC activity (Fig. 2H).

In agreement with the NTPgamma S data, the order of potency of NppNHps at activating AC in membranes expressing Galpha sL was GppNHp > IppNHp > XppNHp, but the potencies of NppNHps were lower than the potencies of the corresponding NTPgamma Ss (Fig. 2, E and I). In membranes expressing Galpha sL-N295, NppNHps stimulated AC in the order of potency XppNHp > GppNHp > IppNHp, but again, maximum AC activities were very low (Fig. 2J). XppNHp increased AC activity in membranes expressing Galpha sL-L227/N295 with ~10-fold higher potency than GppNHp (Fig. 2K). These data fit to the data obtained with XTP/GTP (~20-fold potency difference) (Fig. 2C). IppNHp was virtually inactive in membranes expressing Galpha sL-L227/N295 (Fig. 2K). In contrast to NTPgamma Ss, NppNHps had only minimal inhibitory effects on AC activity in membranes expressing Galpha sL-L227 (Fig. 2, H and L).

Regulation of AC Activity by NTPs, NTPgamma Ss, and NppNHps in Sf9 Membranes Expressing Galpha sS and Galpha sS Mutants-- There were no notable differences in the effects of NTPs, NTPgamma Ss, and NppNHps at Galpha sS and Galpha sS-L212 relative to the corresponding Galpha sL constructs, except that AC inhibition by NTPgamma Ss in membranes expressing Galpha sS-L212 was only marginal (Figs. 2H and 3H). Compared with membranes expressing Galpha sS, basal AC activity in membranes expressing Galpha sS-N280 was lower, indicative for the reduction in GDP affinity of the Galpha sS mutant (Fig. 3, E and F). In contrast to the observations made for the Galpha sL/Galpha sL-N295 couple (Fig. 2, E, F, I, and J), Galpha sS-N280 was as efficacious as Galpha sS in activating AC in the presence of NTPgamma Ss or NppNHps at saturating concentrations (Fig. 3, E, F, I, and J). As was true for Galpha sL-L227/N295 (Fig. 2G), Galpha sS-N280 did not discriminate between GTPgamma S, ITPgamma S, and XTPgamma S (Fig. 3F). In contrast, Galpha sS-N280 exhibited ~20-fold higher potency for XppNHp than for GppNHp/IppNHp (Fig. 3J). Relative to Galpha sS, the potency of XppNHp at Galpha sS-N280 was increased ~240-fold, whereas the potency of GppNHp was reduced ~20-fold (Fig. 3, I and J).


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Fig. 3.   Effects of NTPs, NTPgamma Ss, and NppNHps on AC activity in Sf9 membranes expressing Galpha sS proteins. AC activity in Sf9 membranes was determined as described under "Experimental Procedures." Reaction mixtures contained Sf9 membranes (20-40 µg protein/tube) expressing Galpha sS (A, E, and I), Galpha sS-N280 (B, F, and J), Galpha sS-L212/N280 (C, G, and K), or Galpha sS-L212 (D, H, and L) in the presence of NTPs (A-D), NTPgamma Ss (E-H), or NppNHps (I-L) at the concentrations indicated on the abscissa. Log -10 designates the absence of added guanine, hypoxanthine, or xanthine nucleotide. Note that because of the high AC activities with membranes expressing Galpha sS-L212, the scale of the y axis in D, H, and L is different than from in the other panels. Data were analyzed by nonlinear regression and best fitted to sigmoid concentration-response curves. Data shown are the means ± S.D. of four to six experiments performed in duplicates.

Similar to the observations for the Galpha sL-L227/Galpha sL-L227/N295 couple (Fig. 2, C and D), introduction of the N280 mutation into Galpha sS-L212 decreased basal AC activity (Fig. 3, C and D), indicative for a decrease in GDP affinity. The introduction of the Leu212 mutation into Galpha sS-N280 had considerable impact on the potencies of NTPgamma S and NppNHps. Specifically, XTPgamma S activated Galpha sS-L212/N280 with ~10-fold higher potency than GTPgamma S and ITPgamma S, whereas Galpha sS-N280 did not discriminate between NTPgamma Ss (Fig. 3, F and G). By analogy, the Galpha o, Galpha 11, and Galpha 16 Gln/Leu- Asp/Asn double mutants exhibited XTPgamma S selectivity (5, 12, 13). Moreover, at Galpha sS-L212/N280, IppNHp was only 2-fold less potent than XppNHp, whereas at Galpha sS-N280, IppNHp was ~20-fold less potent than XppNHp (Fig. 3, J and K). We also noted similar NTP potencies at Galpha sS-L212/N280 (Fig. 3C).

Regulation of AC Activity in Sf9 Membranes Expressing beta 2AR plus Galpha s Proteins-- We wished to determine whether Galpha s mutants interact with the beta 2AR. The beta 2AR is constitutively active, i.e. even in the absence of agonist, beta 2AR promotes nucleotide exchange at Galpha s, resulting in stimulatory effects of NTPs on basal AC activity (17, 18). The inverse agonist ICI 118,551 inhibits the effects of agonist-free beta 2AR. In membranes co-expressing beta 2AR and Galpha sS-N280, XTP increased basal AC activity with ~50-fold higher potency than GTP (Fig. 4, A and C). This potency difference fits with the observations made for XppNHp/GppNHp (Fig. 3J) and contrasts with the lack of XTPgamma S selectivity (Fig. 3F). ICI 118,551 abrogated the stimulatory effects of GTP and XTP on basal AC activity in membranes co-expressing beta 2AR and Galpha sS-N280, indicating that the agonist-free beta 2AR stimulated nucleotide exchange at Galpha sS-N280. Additionally, the beta 2AR agonist ISO further increased AC activities in the presence of GTP and XTP (Fig. 4, A and C), supporting the view that Galpha sS-N280 couples to the beta 2AR. Based on the high potency of XTP at Galpha sS-N280 in terms of AC activation, we expected Galpha sS-N280 to exhibit high-affinity XTPase activity. In fact, in membranes expressing beta 2AR-Galpha sS-N280, ISO stimulated steady-state XTP hydrolysis with a Km of 240 ± 80 nM and a Vmax of 0.27 ± 0.02 min-1 (means ± S.D., n = 3). The Km value for GTP hydrolysis was reduced to >4 µM.


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Fig. 4.   Analysis of AC activity in Sf9 membranes expressing beta 2AR plus Galpha s proteins. AC activity in Sf9 membranes was determined as described under "Experimental Procedures." Reaction mixtures contained Sf9 membranes (20-30 µg protein/tube) expressing the beta 2AR plus Galpha sS-N280 (A and C) or the beta 2AR plus Galpha sL-L227/N295 (B and D). Reaction mixtures contained GTP (A and B) or XTP (C and D) at the concentrations indicated on the abscissa in the presence of solvent (basal), 10 µM ISO, or 1 µM ICI 118,551 (ICI). Log -10 designates the absence of added GTP or ITP. Data were analyzed by nonlinear regression and best fitted to sigmoid concentration-response curves. Data shown are the means ± S.D. of four to six experiments performed in duplicates.

Galpha sL-N295 failed to reconstitute beta 2AR-stimulated AC activity with GTP or XTP (data not shown), corroborating the weak functional activity of this mutant. Similar to the data obtained with membranes expressing Galpha sL-L227/N295 alone (Fig. 2C), XTP was severalfold more potent than GTP at activating AC in membranes co-expressing beta 2AR plus Galpha sL-L227/N295 (Fig. 4, B and D). The inverse agonist ICI 118,551 did not inhibit AC stimulation by NTPs, supporting the conclusion that the effects of NTPs were due to their hydrolysis resistance at Galpha sL-L227/N295. ISO strongly reduced basal AC activity in membranes co-expressing the beta 2AR and Galpha sL-L227/N295. A model in which ISO stimulates GDP dissociation from Galpha sL-L227/N295 and increases the abundance of nucleotide-free Galpha sL-L227/N295 explains these data. Nucleotide-free Galpha s is less efficient than Galpha s-GDP at activating AC (22, 23). ISO increased the diminished basal AC activity in the presence of GTP and XTP, indicating that the beta 2AR also stimulated GTP/XTP binding to Galpha sL-L227/N295 (Fig. 4, B and D). However, the AC activities in the presence of ISO always remained well below the activities in the absence of ISO, indicating that nucleotide dissociation dominated nucleotide binding. Finally, beta 2AR-Galpha sL-L227/N295 did not exhibit ISO-stimulated GTPase or XTPase activity (data not shown). Thus, receptor regulation was intact in Galpha sS-N280 but defective in Galpha sL-L227/N295 and Galpha sL-N295.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Role of GDP Affinity in the Function of Xanthine Nucleotide-selective Galpha Proteins-- In small GTP-binding proteins, the Asp/Asn mutation switches the base selectivity from guanine to xanthine and is valuable for the analysis of a specific GTP-binding protein in complex systems (8-11). Similar applications could be devised for xanthine nucleotide-selective Galpha proteins. Because of the conserved guanine nucleotide binding in small GTP-binding proteins and Galpha (3, 7), the achievement of this goal was thought to be straightforward. However, Galpha o, Galpha 11, Galpha 16, and Galpha sL Asp/Asn mutants were inactive or exhibited weak activity at best (Fig. 2, B, F, and J) (5, 12, 13). An explanation for the poor activity of these proteins could be their low GDP affinity, a consequence of the Asp/Asn mutation. Because cellular GDP concentrations are much higher than XDP concentrations (5), Galpha Asp/Asn mutants are likely to exist predominantly in the nucleotide-free state, particularly if GDP affinity of the parent Galpha is low anyway. In fact, Galpha o and Galpha sL possess low GDP affinities (16, 31). However, in the nucleotide-free state, Galpha is instable and denatures (30). Thus, because of their low GDP affinity, Galpha o, Galpha 11, and Galpha sL Asp/Asn mutants may already denature during expression. Denaturation apparently happens at temperatures as low as 28 °C, i.e. the incubation temperature for Sf9 cells. Galpha o, Galpha 11, and Galpha 16 Asp/Asn mutants were expressed at 37 °C (5, 12, 13), further increasing the probability of denaturation.

Galpha sS possesses ~2-3-fold higher GDP affinity than Galpha sL (16). This difference in GDP affinity is too small to result in different basal AC activities in membranes expressing Galpha sS and Galpha sL (Figs. 2A and 3A). Only in the presence of agonist-free beta 2AR, which preferentially stimulates GDP dissociation from Galpha sL, do different basal AC activities with Galpha sS and Galpha sL become evident (22). Apparently, the moderately higher GDP affinity of Galpha sS relative to Galpha sL is sufficient to prevent denaturation of Galpha sS-N280 during expression because Galpha sS-N280 was as effective as Galpha sS at activating AC (Fig. 3, E, F, I, and J). Moreover, Galpha sS-N280 possesses the expected XTP selectivity in terms of AC activation and NTP hydrolysis and is receptor-regulated (Fig. 4, A and C). Compared with Galpha sS, the Asp/Asn mutation increased the potency of XppNHp by ~250-fold (Fig. 3, I and J). This increase in potency equals or even exceeds the increases in xanthine nucleotide potency at Asp/Asn mutants of small GTP-binding proteins (8-11). Accordingly, XppNHp at 1 µM almost maximally activates Galpha sS-N280 without a stimulatory effect on Galpha sS or Galpha sL (Figs. 2I and 3, I and J). These potency differences render XppNHp a highly selective Galpha sS-N280 activator. Thus, to the best of our knowledge, Galpha sS-N280 represents the first fully functional xanthine nucleotide-selective Galpha with the Asp/Asn mutation alone. Accordingly, Galpha sS-N280 provides an excellent model to clarify the still poorly understood role of Galpha sS relative to the role of Galpha sL in signal transduction (16, 22, 24-26, 32).

The requirement for the additional Gln/Leu mutation in Galpha o, Galpha 11, Galpha 16, and Galpha sL Asp/Asn mutants for functionally active xanthine nucleotide-selective Galpha proteins can be explained by the mutation-induced increase in GDP affinity, preventing Galpha denaturation during expression (Fig. 2, C, G, and K) (5, 12-15). The increase in GDP affinity in Galpha sL-L227/N295 relative to Galpha sL-N295 is reflected by the strong increase in basal AC activity (Fig. 2, B and C) and ISO-induced AC inhibition in the absence of GTP or XTP (Fig. 4, B and D). However, the introduction of the Gln/Leu mutation in Galpha sL-N295 increases the GDP affinity and basal AC activity so strongly that the signal-to-noise ratio of this Galpha s mutant is poor, i.e. the stimulatory effects of NTPgamma Ss and NppNHps are much smaller than those with Galpha sS-N280 (Fig. 2, G and K and Fig. 3, F and J). In addition, the high GDP affinity and reduction of NTPase activity in Galpha sL-L227/N295 compromises receptor regulation of this Galpha mutant (Fig. 4, B and D). However, by systematically mutating Gln227 in Galpha sL against other amino acids than leucine or targeting other conserved amino acids that regulate GDP affinity such as Ala366 in Galpha sL (30), it could be possible to adjust the GDP affinity of any Galpha to a level that ensures both functional expression and excellent signal-to-noise ratio. Fortuitously, Galpha sS possesses the optimal GDP affinity to fulfill both prerequisites for a xanthine nucleotide-selective Galpha without the need for mutagenesis.

Perturbation of the Catalytic Site Alters Nucleotide Selectivity of Galpha Proteins-- GTPgamma S, but not GppNHp, sterically perturbs the structure of the catalytic site of Galpha (6). Accordingly, Galpha -GppNHp resembles Galpha -GTP more closely than Galpha -GTPgamma S. Our studies with Galpha mutants support the conclusions of the crystallographic studies. Most strikingly, Galpha sS-N280 and Galpha sL-L227/N295 did not discriminate between NTPgamma Ss, and NTPgamma Ss exhibited ~5-10-fold reduced potencies compared with the potencies of GTPgamma S at Galpha sS and Galpha sL (Figs. 2G and 3F). In contrast to the NTPgamma S data, Galpha sS-N280 and Galpha sL-L227/N295 exhibited selectivity for XTP and XppNHp relative to GTP and GppNHp, respectively (Fig. 2, C and K, Fig. 3J, and Fig. 4, A-D). Thus, steric perturbation of the catalytic site of Galpha s Asp/Asn mutants by NTPgamma Ss annihilates xanthine nucleotide selectivity. Apparently, perturbation of the catalytic site propagates a conformational change in Galpha s, reorienting Asn280/Asn295, so that these amino acids do not participate in hydrogen bonding of NTPgamma Ss anymore. As consequences of the functional neutralization of Asn280/Asn295, base selectivity is lost, and NTPgamma S affinity of Galpha s mutants is reduced. Thus, to take advantage of the xanthine nucleotide selectivity of Galpha sS-N280 and Galpha sL-L227/N295, it is crucial to use XppNHp or XTP but not XTPgamma S. Based on all these findings, we predict that the crystal structures of the catalytic site of Galpha -Asp/Asn-XppNHp/XTP resemble the corresponding structures of Galpha -GppNHp/GTP. Moreover, the crystal structures of the catalytic site of Galpha -Asp/Asn-XTPgamma S and Galpha -GTPgamma S should be similar.

The Gln/Leu mutation perturbs the structure of the catalytic site as well (6). In Galpha sL, the Gln/Leu mutation converted NTPgamma Ss from activators into inhibitors and abolished base selectivity (Fig. 2H). The Gln/Leu mutation also profoundly changed the interactions of Galpha sS-N280 with nucleotides. Most notably, at Galpha sS-L212/N280, XTPgamma S was ~10-fold more potent than GTPgamma S, whereas at Galpha sS-N280, GTPgamma S and XTPgamma S were similarly potent (Fig. 3, F and G). Thus, a combination of two perturbing factors in the catalytic site (NTPgamma S and Gln/Leu mutation) revealed xanthine nucleotide selectivity of Galpha sS-L212/N280. One can envisage that the conformational change induced by the Gln/Leu mutation is functionally compensated by the NTPgamma S-induced conformational change, leaving the orientation of Asn280 intact and preserving XTPgamma S selectivity of Galpha sS-L212/N280. Such a process also apparently took place in the Gln/Leu-Asp/Asn double mutants of Galpha o, Galpha 11, and Galpha q (5, 12, 13). In contrast to NTPgamma Ss, NppNHps and NTPs should not neutralize the conformational change induced by the Gln/Leu mutation in Galpha sS-N280. In fact, xanthine nucleotide selectivity for NTPs and NppNHps in Galpha sS-L212/N280 is substantially reduced relative to Galpha sS-N280 (Fig. 3C, J, and K and Fig. 4, A and C).

Conclusions-- Galpha sS-N280 is a fully functional xanthine nucleotide-selective Galpha and provides an excellent model to study the specific roles of Galpha sS in signal transduction. A sufficiently high GDP affinity of Galpha Asp/Asn mutants is crucial for expression of functionally active proteins. The order state of the catalytic site of Galpha critically determines nucleotide-Galpha interactions. Specifically, NTPgamma Ss and the Gln/Leu mutation perturb the catalytic site, but in the case of Galpha sS-L212/N280, the combination of two perturbing factors actually results in xanthine nucleotide selectivity. Moreover, our data show that XTPgamma S and XppNHp are not functionally equivalent activators of Galpha Asp/Asn mutants, and caution must be exerted when making the decision which nucleotide to use. Furthermore, our data explain some as yet poorly understood properties of xanthine nucleotide-specific Galpha mutants (5, 12, 13). We anticipate that functionally active xanthine nucleotide-selective Galpha mutants with high signal-to-noise ratio can be generated for any given Galpha , but an individualized mutagenesis approach for each Galpha has to be taken. The availability of an array of xanthine nucleotide-selective Galpha mutants will complement gene knockout approaches to study the functions of individual Galpha proteins in intact cell systems.

    ACKNOWLEDGEMENTS

We thank Dr. H.-Y. Liu for conducting some preliminary studies for this project and Dr. S. R. Sprang (Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX) for stimulating discussions.

    FOOTNOTES

* This work was supported by Grant 0051404Z of the Heartland Affiliate of The American Heart Association (to R. S.), the J. R. & Inez Jay Biomedical Research Award of The University of Kansas (to R. S.), and a predoctoral fellowship of the Studienstiftung des Deutschen Volkes (to A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is in remembrance of the 12th anniversary of the reunification of Germany on October 3rd, 2002, without which this project would not have been conducted.

|| To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, The University of Kansas, Malott Hall, Rm. 5064, 1251 Wescoe Hall Dr., Lawrence, KS 66045-7582. Tel.: 785-864-3525; Fax: 785-864-5219; E-mail: rseifert@ku.edu.

Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M210162200

    ABBREVIATIONS

The abbreviations used are: Galpha , unspecified G-protein alpha -subunit; AC, adenylyl cyclase; beta 2AR, beta 2-adrenoceptor; beta 2AR-Galpha s, fusion protein containing the beta 2AR and a Galpha s protein; Galpha sL, long splice variant of Galpha s; Galpha sS, short splice variant of Galpha S; Galpha sL-L227, Galpha sL mutant with a Q227L exchange; Galpha sL-N295, Galpha sL mutant with a D295N exchange; Galpha sL-L227/N295, Galpha sL mutant with a Q227L- and D295N exchange; Galpha sS-L212, Galpha sS mutant with a Q212L exchange; Galpha sS-N280, Galpha sS mutant with a D280N exchange; Galpha sS-L212/N280, Galpha sS mutant with a Q212L- and D280N exchange; GppNHp, guanosine 5'-[beta ,gamma -imido]- triphosphate; GTPgamma S, guanosine 5'-[gamma -thio]triphosphate; IppNHp, inosine 5'-[beta ,gamma -imido]triphosphate; ISO, (-)-isoproterenol; ITPgamma S, inosine 5'-[gamma -thio]triphosphate; NppNHp, nucleoside 5'-[beta ,gamma -imido]triphosphate; NTP, nucleoside 5'-triphosphate; NTPgamma S, nucleoside 5'-[gamma -thio]triphosphate; XppNHp, xanthosine 5'-[beta ,gamma -imido]triphosphate; XTP, xanthosine 5'-triphosphate; XTPgamma S, xanthosine 5'-[gamma -thio]triphosphate; IDP, inosine 5'-diphosphate; XDP, xanthosine 5'-diphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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