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Albumin: a Gαs-specific guanine nucleotide dissociation inhibitor and GTPase activating protein
ABB Archives of Biochemistry and Biophysics 415 (2003) 221–228 www.elsevier.com/locate/yabbi
Albumin: a Gas -specific guanine nucleotide dissociation inhibitor and GTPase activating protein Ziyun Du and Tarun B. Patel* Department of Pharmacology, University of Tennessee, The Health Science Center, 874 Union Avenue, Memphis, TN 38163, USA Received 19 March 2003, and in revised form 14 May 2003
Abstract Heterotrimeric GTP binding protein (G protein)-mediated signal transduction events are regulated by their effectors and regulators of G protein signaling (RGS) protein family. The latter proteins function as GTPase activating proteins (GAPs) for G protein a subunits and terminate signaling events. In a search for proteins that modulate the activity of the stimulatory G protein of adenylyl cyclase (Gas ), we found that bovine serum albumin (BSA) inhibits the steady-state GTPase activity of Gas , but not the inhibitory G protein (Gai1 ). This effect of BSA is mediated by decreasing the rate of GDP dissociation from Gas and decreasing the rate of GTP binding. Thus, BSA functions as a guanine nucleotide dissociation inhibitor for Gas . Moreover, BSA also increased the intrinsic GTPase activity of Gas , but not Gai1 , demonstrating that BSA functions as a Gas -specific GAP. Using mutants of Gas (Q227L, Q227N, R201C, and R201K), we demonstrate that BSA mediates its GAP function by modulating the ability of R201 to increase GTPase activity. Moreover, using wild-type and Q227N forms of Gas , our studies demonstrate that the GDI function of BSA decreases the ability of Gas to stimulate adenylyl cyclase. These findings assign a novel function to BSA as a regulator of G protein signaling. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: G proteins; Adenylyl cyclase; Bovine serum albumin; GTPase activity; GTPase activating protein; Guanine nucleotide dissociation inhibitor; Cyclic AMP; Stimulatory GTP binding protein of adenylyl cyclase
Heterotrimeric GTP binding proteins (G proteins1) are activated by heptahelical transmembrane receptors upon binding of their respective ligands such as hormones, neurotransmitters, and chemokines (reviewed in [1–3]). The ligand-bound, active receptors increase the exchange of GDP for GTP on the Ga subunits of the G proteins and the GTP-bound Ga subunit dissociates from the Gbc dimer. The active, GTP-bound, Ga subunits *
Corresponding author. Present address: Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA. Fax: 1-708-216-6888. E-mail address: [email protected] (T.B. Patel). 1 Abbrevations used: G protein, GTP binding protein, Gas and Gai1 , a subunits of the stimulatory and inhibitory GTP binding proteins of adenylyl cyclase, respectively; BSA, bovine serum albumin; RGS, regulators of G protein signaling; GAP, GTPase activating protein; GDI, guanine nucleotide dissociation inhibitor; DTT, dithiothreitol; GTPcS, guanosine-50 -O-(3-thiotriphosphate); C1–C2 ACV, soluble, recombinant, type V adenylyl cyclase comprising of the cytosolic domains.
subsequently regulate the downstream effectors such as adenylyl cyclase with a resultant increase (Gas ) or decrease (Gai ) in cAMP production (reviewed in [1–3]). The intrinsic GTPase activity of the Ga subunits hydrolyzes the bound GTP and the Ga subunit returns to its inactive GDP-bound state. Regulators of G protein signaling (RGS) proteins that increase the intrinsic GTPase activity of Ga subunits facilitate the inactivation of Ga subunits (reviewed in [4,5]). Therefore, the RGS proteins are also referred to as GTPase activating proteins (GAPs). To date, more than 20 RGS protein family members have been identified ([4,5] for reviews). Several of the RGS proteins are GAPs for Gi , Gz Gq , or G12=13 classes of G proteins [4,5]. However, recently, a new member of the RGS family, RGS-PX1, was also reported to act as a GAP for Gas [6]. In addition to the RGS proteins, some effectors such as PLC and adenylyl cyclase also function as GAPs for Gaq and Gas , respectively [7,8]. Bovine serum albumin (BSA), one of the most abundant proteins in the blood is synthesized and
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00263-7
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secreted by liver cells and plays a key role in the transport of fatty acids, metabolites, and drugs as well as in maintaining colloidal osmotic pressure [9,10]. Because no enzymatic or other functional activity has been identified for BSA, it has been used in numerous biochemical studies as an inert, control, protein against which active proteins are tested. Alternatively, BSA has been used to increase the protein content of samples to stabilize other proteins or protect them from proteolysis. We utilized BSA as a control protein in a screen for proteins that modulate Gas activity. Unexpectedly, we observed an inhibitory effect of BSA on the steady-state GTPase activity of Gas , without any effect on steadystate GTPase activity of Gai . These actions of BSA were obliterated by heat denaturation or trypsin digestion. Moreover, we observed that BSA acts as a specific GAP for Gas . Additionally, experiments with mutant forms of Gas showed that BSA may exert its GAP actions by modulating the ability of R201 to stabilize the oxygens on the phosphoryl (c phosphate of GTP) leaving group. Finally, we report that BSA, by regulating the activity of Gas , alters the ability of the G protein to activate adenylyl cyclase.
Materials and methods Fatty acid-free bovine albumin (fraction V; >98% pure, catalog number 152401) was purchased from ICN Biomedicals (Aurora, OH). This is the only commercially available form of albumin that is essentially free of fatty acids (<0.05 mg/g protein). Likewise, fraction V of human albumin was purchased from Sigma Chemical (St. Louis, MO; catalog number A-1653). All other chemicals were of the highest purity available from commercial sources. Recombinant Ga subunits Recombinant bovine Gas (unmodified or with Nterminus His X 6 tag) and myristoylated rat Gai1 were expressed and purified, as previously described [11–13]. The Gas used in our experiments was the short form (380 amino acid long). However, for convenience, the amino acid numbering system used is that for the long form (394 amino acids) of Gas . Mutations of Q227 to L or N, and R201 to C or K were introduced into wild type construct using mutagenic oligodeoxynucleotides and universal polymerase chain reaction. Soluble form of type V adenylyl cyclase The plasmid construct to express soluble type V adenylyl cyclase, comprising the C1 and C2 cytoplasmic domains (C1–C2 ACV) joined by an artificial linker, has
been previously described [12]. The C1–C2 ACV was expressed in TP2000 strain of Escherichia coli at 37 °C and inclusion bodies were isolated and solubilized with 6 M guanidinium hydrochloride. The denatured protein (20 lg/ml) was then refolded by dialysis (three times for 10 h each) at 4 °C against 10 volumes of buffer containing 1/10 (v/v) of PBS, pH 8.0, 10 mM sodium pyrophosphate, 100 lM ATP, 1 mM DTT, 100 lM MgCl2 , and 10% glycerol. The dialyzed protein was concentrated using Centricon Plus 80 (10,000 MW cut-off; Millipore, Bedford, MA) and buffer exchanged to 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, and 20% (v/v) glycerol. The protein at this stage was approximately 10–20% pure but demonstrated significant adenylyl cyclase activity. Steady-state GTPase activity Steady-state GTPase activity was measured in 100 ll of buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgCl2 , 1 mM DTT, 1 lM [c-32 P]GTP (5000 dpm/ pmol), and the indicated concentration of Gas or Gai1 in the presence and absence of BSA at room temperature for 10–20 min. The reactions were terminated with icecold charcoal slurry (5% (w/v) in 50 mM NaH2 PO4 ) and after centrifugation (20,000g for 10 min) aliquots of the supernatant were counted for 32 Pi release. GTPase activating protein assays The single cycle GTPase activity was measured as described by Berman et al. [14] with the following modification: Gas (2.0 lM) was first incubated at room temperature for 10 min in 50 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, and 1 lM [c-32 P]GTP (60 dpm/fmol). The temperature was lowered to 4 °C and incubation was continued for another 10 min. For time zero, 10 ll aliquots (20 pmol) were withdrawn and quenched with 0.99 ml of charcoal suspension in 50 mM NaH2 PO4 described above. Within 10 s of withdrawing the zero time point, 100 ll of the reaction mixture was diluted to 1 ml with buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgCl2 , 1 mM DTT, and 1 mM GTP. At the indicated time points, 100 ll aliquots were withdrawn and mixed with 0.9 ml of charcoal suspension. 32 Pi release was monitored as described for steady-state GTPase activity assay. GDP release and GTPcS binding GDP release was measured as follows: Gas (2.0 lM) was loaded with trace amount of [a-32 P]GTP in buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgSO4 , and 1 mM DTT for 1 h at room temperature to ensure the complete hydrolysis of bound GTP to GDP. The reaction mixture was then diluted 10 times in a buffer con-
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taining 50 mM Tris–HCl, pH 8.0, 5 mM MgSO4 , 1 mM DTT, and 1 mM GTP. Aliquots (100 ll; 20 pmol Gas ) were withdrawn at the indicated times and filtered through protran BA85 filters (Schleicher and Schuell, Keene, NH), as described previously [11]. GTPcS binding was measured by incubating Gas (200 nM) at room temperature in buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgSO4 , 1 mM DTT, and 1 lM [35 S]GTPcS (5000 dpm/pmol). At the indicated times, 100 ll aliquots were withdrawn and filtered as described above for GDP release experiments. Adenylyl cyclase activity assays Adenylyl cylase activity was measured as described previously [12] with 40 pmol of soluble C1–C2 ACV and 41 pmol Gas in 100 ll reaction mix containing 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2 , 10 lM GTP or GTPcS, 1 mM of 3-isobutyl-1-methylxanthine, 0.1 mM ATP, 1 mM cAMP, 15,000 dpm [3 H]cAMP, and 400 dpm/ pmol [a-32 P]ATP in the presence or absence of BSA at room temperature for 20 min. The ATP regenerating system was omitted from the assay mixture so that GDP resulting from the GAP function of BSA would not be converted back to GTP. Reactions were initiated by adding the Gas and were stopped with 50 ll ice-cold, 3 stopping buffer (150 mM Tris–HCl, pH 7.4, 6% SDS, 3 mM cAMP, and 3 mM ATP). Cyclic AMP was separated by chromatography on two sequential columns of Dowex AG50 WX-4 and alumina and the amount of 32 P label in this fraction was determined by liquid scintillation counting [15].
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Results and discussion In a screen for proteins that altered Gas activity, we included BSA as a control and monitored its ability to modulate the steady-state GTPase activity of Gas . Surprisingly, BSA in a concentration-dependent manner inhibited the steady-state activity of Gas (Fig. 1A). Interestingly, BSA did not alter the steady-state GTPase activity of Gai (Fig. 1A), indicating that the effect on Gas was specific. Consistent with this latter notion, it has also been demonstrated that BSA does not have any effect on steady-state GTPase activity of transducin (Gat ) [16]. Together, these findings suggest that the inhibitory effect of BSA on steady-state GTPase activity of Gas is not due to non-specific interactions, but rather a specific interaction between Gas and BSA. BSA inhibited the steady-state GTPase activity of both N-terminal hexahistidyl-tagged or untagged Gas (data not shown), indicating that N-terminal modifications of Gas do not alter its interactions with BSA. To rule out the possibility that non-protein contaminants in the BSA may be inhibiting the steady-state GTPase activity of Gas , the experiments depicted in Fig. 1B were performed. Denaturation of BSA by heating or digestion of BSA with trypsin obliterated the ability of BSA to inhibit the steady-state GTPase activity of Gas (Fig. 1B); in the latter approach after digestion of BSA, trypsin was inactivated by heating (10 min at 100 °C). Heat inactivated trypsin, by itself, did not alter the steady-state GTPase activity of Gas (Fig. 1B). Fig. 1C shows that under the experimental conditions of Fig. 1B, trypsin almost completely digested the BSA. Note that the Coomassie
Fig. 1. Inhibition of the steady-state GTPase activity of Gas by BSA. Steady-state GTPase activities were measured for 10 min at room temperature as described in Materials and methods with either 200 nM each of Gas () or Gai1 () in presence of the indicated BSA concentrations (A). (B) Same as (A) with 200 nM of Gas except that BSA was treated as follows. BSA (300 lM) was either left untreated (BSA), or heat denatured (heated BSA) or digested with 100 lg/ml trypsin (BSA + trypsin) overnight at room temperature in buffer containing 50 mM Tris–HCl, pH 8.0, 1 mM DTT. As an additional control, 100 lg/ml trypsin without BSA (trypsin alone) was also incubated for the overnight period. Thereafter, where applicable, trypsin was inactivated by heating (100 °C for 10 min). The control and experimental samples were then centrifuged (20,000g, 10 min) and aliquots of supernatants that would yield 30 lM equivalent final concentration of BSA or 10 lg/ml trypsin were used. The effect of human serum albumin (HSA; 30 lM) on steady-state GTPase activity is also shown. (C) BSA was treated with and without trypsin as described under (B) above and 20 lg of the protein was applied to SDS–PAGE. The Coomassie stained gel shows that trypsin nearly completely digested the BSA (lane 3). Representatives of at least two experiments performed in duplicate are shown.
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stained gel (Fig. 1C) does not show any other protein bands, indicating that as reported by the manufacturer (ICN Biomedicals), the BSA is >98% pure. Together, the findings in Figs. 1B and C demonstrate that BSA, and not some non-protein contaminant, alters the steady-state GTPase activity of Gas . Human serum albumin that shares about 80% sequence homology with BSA also inhibited the steady-state GTPase activity of Gas (Fig. 1B), demonstrating the generality of the effects among albumins. The specific inhibition of steady-state GTPase activity of Gas by BSA prompted us to perform additional experiments to understand the mechanism of inhibition and its significance on the production of cAMP. The steady-state GTPase activity of Gas is determined by the rate of GTP–GDP exchange [17]. Thus, this activity could be modulated either by the inhibition of GDP release, the rate-limiting step in the steady-state GTP hydrolysis of Gas , and/or the inhibition of GTP hydrolysis per se. Therefore, first, we investigated whether BSA altered the rate of guanine nucleotide exchange on Gas . The rate of GTP–GDP exchange on Ga subunits can be monitored by following either the binding of GTPcS or the release of bound GDP. As shown in Fig. 2A, BSA decreased the rate of GTPcS binding to Gas such that the t1=2 for maximal GTPcS binding to Gas increased from 5.3 0.8 min in the absence of BSA to 14.3 0.6 min in its presence. Likewise, BSA also decreased the rate of GDP dissociation from Gas (Fig. 2B). These findings demonstrate that BSA acts as a guanine nucleotide dissociation inhibitor (GDI) for Gas and thereby contributes to the inhibition of steadystate GTPase activity of the G protein. To determine whether BSA also altered the intrinsic GTPase activity of Gas per se, the experiments depicted in Fig. 3 were performed. To monitor GTPase activity without any interference from alterations in the rate of guanine nucleotide exchange, we measured the single
Fig. 2. BSA functions as a guanine nucleotide dissociation inhibitor (GDI) for Gas . GTPcS binding (A) and GDP release (B) in the absence () or presence () of 30 lM BSA were assayed as described in Materials and methods. Total amount of Gas was 1 lg (20.5 pmol) in 100 ll of reaction mix. From the maximal binding of GTPcS (10 pmol), it was estimated that the Gas was 50% active. Representatives of two similar experiments performed in duplicate are shown.
Fig. 3. BSA accelerates the intrinsic GTPase activity of Gas , but not of Gai1 . The single cycle GTPase activities were assayed as described in Materials and methods with Gas (20.5 pmol protein, A) or Gai1 (25 pmol protein, B) in the absence () or presence () of 30 lM BSA. Gas and Gai1 were loaded with [c-32 P]GTP at room temperature and the hydrolysis was carried out at 4 °C for Gas and at room temperature for Gai1 , respectively. Representatives of at least two similar experiments are shown.
cycle GTP hydrolysis of Gas . For this purpose, Gas was first loaded with [c-32 P]GTP in presence of EDTA to block the hydrolysis of the bound GTP [14]. After incubation, the GTP bound Gas was diluted with buffer containing 1000 times excess amount of unlabeled GTP in presence of Mg2þ to initiate the hydrolysis of the bound [c-32 P]GTP in the presence and absence of BSA. This assay measures hydrolysis of the pre-bound [c32 P]GTP without having to rely on further GTP binding. As shown in Fig. 3A, BSA markedly accelerated the single cycle GTP hydrolysis of Gas but had no effect on the single cycle GTP hydrolysis of Gai1 (Fig. 3B). It has also been reported that BSA, at the concentrations used in our assays, does not affect the single cycle GTP hydrolysis of transducin (Gat ) [16]. Thus, like the GDI effect (Figs. 1 and 2), the GTPase activating property of BSA is also specifically directed at Gas . The t1=2 for complete GTP hydrolysis by Gas in the single cycle GTPase assay is about 1 min at 0 °C in the absence of BSA (Fig. 3A), which is same as that reported by Berman et al. [14], but 5 times faster than that reported by Zheng et al. [6]. However, in the presence of BSA, all of the bound GTP was hydrolyzed before the first time point (15 s) could be withdrawn (Fig. 3A). Because of the rapid GTP hydrolysis, we were unable to accurately estimate the t1=2 of complete GTP hydrolysis in the single cycle GTPase assay in presence of BSA; a conservative estimate would suggest that the intrinsic GTP hydrolysis rate of Gas in presence of BSA is at least 10 times faster than that of Gas in absence of BSA. Overall the data in Fig. 3 demonstrate that BSA increases the intrinsic GTPase activity of Gas and in this respect acts as a GTPase activating protein (GAP). Thus, just as RGS proteins act as GAPs for Gai , Gaz; and Gaq [4,5], BSA acts as a GAP for Gas but not for Gai (Fig. 3B) or Gat [16]. It has been shown that RGS4, a GAP for Gai1 , has no effect on the intrinsic GTPase activity of Gai1 Q204L
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mutant, but stimulates the GTPase activity of its R178C mutant [14]. The cognate conserved glutamine (Q227) and arginine (R201) residues on Gas are also important in regulating its GTPase activity [18,19]. Therefore, we determined whether mutations of Q227 or R201 on Gas would modulate the GDI or GAP activities of BSA. First, we determined the steady-state GTPase activity of the constitutively active Gas mutants Q227L, Q227N, R201C, and R201K. Consistent with previous findings [18,19], these mutants have a low GTPase activity (Fig. 4A). Thus, Q227L and R201C mutants of Gas exhibited less than 5% of the wild type steady-state GTPase activity (Fig. 4). Likewise, the steady-state GTPase activity of Q227N and R201K mutants was approximately 10% and 30% that of the wild type protein, respectively (Fig. 4A). Because of the low intrinsic GTPase activity, the effects of BSA, if any, on the steady-state GTPase activities of the mutants were difficult to decipher (Fig. 4A). However, like the wild-type Gas , BSA decreased the release of GDP from all of the Gas mutants, except the R201K form ( 6 10% decrease) (Fig. 4B). The precise reason(s) for the lack of an effect of BSA on GDP release from R201K Gas is presently not apparent since the GDP release from the R201C mutant was inhibited by BSA (Fig. 4B). Overall, however, it would appear that the GDI function of BSA was not altered by mutations of R201 and Q227 to cysteine and leucine or asparagine, respectively. The GTPase activity of Q227L and R201C mutants of Gas is very low and BSA did not alter the single cycle GTP hydrolysis of these mutants (Figs. 5A and C). This is in contrast to the effects of RGS4 on the equivalent mutants of Gai [14]. In this latter study, RGS4 stimu-
Fig. 4. BSA does not modulate the steady-state GTPase activity of mutant forms of Gas , but inhibits GDP release from these mutants. (A) The steady-state GTPase activities of Gas mutants were measured with 20.5 pmol each of the proteins in 100 ll of reaction mixture in the absence (open bar) or presence (filled bar) of 30 lM BSA as described in Fig. 1 and Materials and methods. (B) GDP release was measured as described in Materials and methods. Wild-type and mutant forms of Gas (20.5 pmol each) were incubated in a final volume of 10 ll with trace amount of [a-32 P]GTP in the presence of MgSO4 at room temperature for 1 h, diluted 10 times with buffer containing 1 mM GTP with or without 30 lM BSA, and incubated further for 15 min. The amount of 32 P labeled nucleotide bound to the protein was determined by filtration. Representatives of at least two similar experiments are shown.
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Fig. 5. BSA stimulates intrinsic GTPase activity of Q227N and R201K mutants, but not the Q227L and R201C mutants, of Gas . The single cycle GTPase activities were measured as described in legend to Fig. 2 and in Materials and methods. Gas mutants (20.5 pmol protein each) were loaded with [c-32 P]GTP at room temperature and the single cycle GTP hydrolysis in 100 ll reaction mixture was monitored at room temperature. (A) Q227L, (B) Q227N, (C) R201C, and (D) R201K in the absence () or presence () of 30 lM BSA. Representatives of at least two similar experiments are shown.
lated single cycle GTPase activity of R178C Gai without altering the activity of Q204L form of Gai [14]. Interestingly, however, the rate of single cycle GTP hydrolysis of the Q227N mutant of Gas was accelerated by at least 50 times to mimic the wild type protein (c.f. Figs. 5B and 3A). Likewise, the single cycle GTPase activity of the R201K mutant of Gas was also slightly augmented in the presence of BSA (Fig. 5D). Together, these results suggest that the stimulation of single cycle GTP hydrolysis of Gas by BSA is probably mediated through alterations in conformation that permit the Q227 and R201 residues to more efficiently hydrolyze the GTP; the R201 residue in heterotrimeric G protein a subunits stabilizes the c-phosphate oxygens and the phosphoryl leaving group [20,21]. These findings are different to those reported for the effects of RGS4 on Q204L and R178C mutants of Gia1 [14] and suggest that BSA and RGS may accelerate the intrinsic GTPase activity of Ga subunits through different mechanisms. The GDI and GAP actions of BSA on Gas would ultimately reduce the amount of the G protein in its active, GTP-bound, state and should decrease the ability of Gas to stimulate cAMP production by adenylyl cyclase. To test the functional significance of BSA-mediated regulation of Gas , we assessed the ability of Gas to
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stimulate the activity of partially purified, recombinant, soluble type V adenylyl cyclase (C1–C2 ACV) in the absence or presence of BSA. We have previously shown that the soluble, recombinant, C1–C2 ACV is regulated by G proteins like its full-length counterpart [12,22]. Thus, the use of soluble C1–C2 ACV permits the evaluation of G protein activity without interference from endogenous Gas that would be present in membranes of mammalian cells. In our approach, using the wild type and Q227N mutant of Gas , we monitored adenylyl cyclase activity in the presence and absence of GTP or GTPcS. The rationale in these experiments was that any effect of BSA on GTP-stimulated AC activity could be due to GDI as well as GAP functions of BSA. However, the effects of BSA on GTPcS-stimulated activity would reflect only the GDI function of BSA since GTPcS is not hydrolyzed by the G protein. The nucleotides were added to the assay mix and reactions were initiated by addition of Gas . As shown in Fig. 6A, wild-type Gas in the presence and absence of GTP did not alter adenylyl cyclase activity. Similar observations with wild-type Gas and GTP have previously been reported [19]. Moreover, BSA did not alter the activity of adenylyl cyclase in the presence of wild-type Gas with or without GTP in the assay (Fig. 6A). However, BSA inhibited by approximately 30% the GTPcS-stimulated adenylyl cyclase activity in the presence of wild type Gas (Fig. 6A). Likewise, the ability of GTPcS to stimulate adenylyl cyclase activity in the presence of Q227N Gas was also inhibited by BSA (Figs. 6A and B). These findings show that the decreased guanine nucleotide exchange on Gas in the presence of BSA (Figs. 2 and 4B) results in diminished ability of the G protein to stimulate adenylyl cyclase. Since GTP did not significantly stimulate the activity of adenylyl cyclase in the presence of wild-type Gas , we could not investigate the GAP function of BSA
using the wild-type Gas . On the other hand, because the Q227N form of Gas activates adenylyl cyclase in the presence of GTP (Fig. 6B) and because BSA significantly stimulates the GTPase activity of this mutant (Fig. 5B), it would be predicted that the ability of Q227N Gas to stimulate adenylyl cyclase activity in the presence of GTP would be markedly inhibited. However, as shown in Fig. 6B, BSA inhibited Q227N Gas stimulated adenylyl cyclase activity in the presence of GTP to the same extent as that observed with GTPcS. The residual activity was surprisingly greater than that observed with Q227N in the absence of GTP (Fig. 6B). These data suggest that either the GAP function of BSA is inconsequential in terms of regulation of adenylyl cyclase or that the hydrolyzed GTP was still bound to the G protein and the GDP along with the bound phosphate mimicked the actions of GTP. To investigate the latter possibility, we measured the binding of 32 P label from [c-32 P]GTP to wild type and Q227N forms of Gas . The binding of the nucleotide was performed in the presence of EDTA as described for the single cycle GTPase activity assays. Binding of [c-32 P]GTP was monitored just prior (0 min) to the addition of MgSO4 with or without BSA. Five minutes after addition of MgSO4 , 32 P label bound to Gas was monitored again. The premise in these experiments was that 5 min after the addition of MgSO4 either in the presence or absence of BSA, the GTP bound to the protein should be completely hydrolyzed (see Figs. 3A and 5B). Indeed, as shown in Fig. 6C, incubation for 5 min with MgSO4 in the presence or absence of BSA decreased the amount of 32 P label associated with wild type Gas . However, the binding 32 P label associated with Q227N Gas was not altered by incubation for 5 min with MgSO4 in the presence or absence of BSA (Fig. 6C). Since the addition of MgSO4 and BSA to Q227N Gas results in a very
Fig. 6. The GDI, but not the GAP, activity of BSA inhibits Gas -stimulated adenylyl cyclase activity. Adenylyl cyclase activities were measured as described in Materials and methods with 3 lg (40 pmol) C1–C2 ACV and 2 lg each (41 pmol) of Gas (wild type or Q227N mutant) in 100 ll of reaction mix in the absence (open bars), and presence (filled bars) of 30 lM BSA. (A) Experiments with wild-type Gas . (B) Same as (A), except that the Q227N Gas was preincubated for 1 h at room temperature in the presence of Mg2þ to ensure that the bound GTP on Gas was hydrolyzed. Reactions were initiated by the addition of Gas and terminated after 20 min. Data presented are means SEM of three determinations. (C) Wild type or Q227N Gas (20.5 pmol each) was incubated for 20 min at room temperature in 10 ll buffer containing 50 mM Tris–HCl, pH 8.0, 1 mM DTT, 1 lM [c-32 P]GTP, and 5 mM EDTA. Thereafter, the reactions were diluted 10 times with buffer containing 50 mM Tris–HCl, pH 8.0, 1 mM DTT, and 5 mM MgSO4 with or without 30 lM BSA. After 5 min at room temperature, the 32 P label associated with Gas was measured by filtration as described in Materials and methods. Means SE (n ¼ 3) are presented.
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rapid hydrolysis of the bound [c-32 P]GTP (Fig. 5B), the data in Fig. 6C strongly suggest that the release of the hydrolyzed phosphate from the Q227N Gas is very slow and therefore the GDP along with the bound phosphate mimics the actions of GTP. In this context, it should be noted that the experiments in the single cycle GTPase assays are terminated with an acidic slurry of charcoal and the denaturation of the G protein permits the release of 32 Pi. Overall, therefore, measurements of the functional significance of the Gas -specific GAP function of BSA remain problematic, even with the use of Q227N mutant of the G protein and the BSA-elicited inhibition of GTP-stimulated adenylyl cyclase activity with this mutant essentially represents the GDI function of BSA. In addition to showing that BSA functions as both GDI and GAP specifically for Gas , in this communication, we also report the identification of a mutant (Q227N) of Gas that is ideally suited to monitor the GAP function of proteins at room temperature. As shown in Fig. 5, the Q227N mutant permits convenient monitoring of GAP activity of proteins at room temperature. Additionally, our data show that the Q227N mutant of Gas has a high affinity for the c-phosphate of the hydrolyzed GTP and the G protein bound GDP and phosphate can mimic the GTP-bound form of the G protein and activate adenylyl cyclase. Presently, it is unclear what the physiological role and consequences of the GDI and GAP function of BSA in vivo would be. Albumin is synthesized and secreted mainly by hepatocytes [23]. However, other cells and organs such as heart, kidney, retina, and pancreas also synthesize albumin [24,25]. Since the secretory process involves vesicular trafficking [26] and because cAMP regulates transcytosis and apical secretion [27] it is tempting to speculate that BSA, by regulating cAMP production, could modulate its own secretion. However, since albumin is contained within vesicles, Gas and albumin may not normally interact with each other. Whether albumin interacts with Gas in some pathological state when albumin is not confined within vesicles is not presently known. In this context, it should be noted that the newly discovered RGS-PX1 that acts as a Gas -GAP [6] is located in endosomes and it has been suggested that RGS-PX1, by altering Gas activity, may play a role in endocytosis or recycling of proteins [6,28]. Albumin secretion from the liver is dysregulated in some chronic conditions such as diabetes, kidney disease, and cancer [10,29,30]. Whether the changes in albumin secretion are also reflected in regulation of cAMP accumulation and downstream alterations in metabolic and other biological events associated with these disease states remains to be determined. Albumin has been reported to be taken up by cells and in some cell types this results in cellular apoptosis [31,32]. Whether the induction of apoptosis by albumin involves modulation of Gas and cAMP generation remains to be determined. The transcellular
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transport of albumin in endothelial cells is mediated by a cell surface glycoprotein (gp60) that has been shown to signal vesicular trafficking via Gai and Src kinase [33]. Thus, while gp60 may activate Gai , its binding partner albumin modulates Gas activity. At the level of adenylyl cyclase, these opposing actions would reinforce each other to decrease cAMP formation. Again, this would require that albumin under some pathological states either leaks out or is released from the vesicles. Moreover, since Gas has also been reported to activate the Src family kinase Lck [34], it is possible that by modulating Gas activity, albumin may regulate the levels of both cAMP and/or the Lck signaling networks. Our findings that BSA acts as a Gas -specific GDI and GAP certainly warrant such investigations. In sum, our findings reported here assign novel functions to BSA and also show that BSA is not necessarily the inert protein that it is assumed to be.
Acknowledgments This work was supported by Grant HL59679 from the NIH (to T.B.P.) and a postdoctoral fellowship (to Z.D.) from the American Heart Association, SE Affiliate. We thank Dr. Helen Poppleton for reading the manuscript and helpful comments. We also thank Mr. David Neblett for technical assistance with this work.
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Albumin: a Gas -specific guanine nucleotide dissociation inhibitor and GTPase activating protein Ziyun Du and Tarun B. Patel* Department of Pharmacology, University of Tennessee, The Health Science Center, 874 Union Avenue, Memphis, TN 38163, USA Received 19 March 2003, and in revised form 14 May 2003
Abstract Heterotrimeric GTP binding protein (G protein)-mediated signal transduction events are regulated by their effectors and regulators of G protein signaling (RGS) protein family. The latter proteins function as GTPase activating proteins (GAPs) for G protein a subunits and terminate signaling events. In a search for proteins that modulate the activity of the stimulatory G protein of adenylyl cyclase (Gas ), we found that bovine serum albumin (BSA) inhibits the steady-state GTPase activity of Gas , but not the inhibitory G protein (Gai1 ). This effect of BSA is mediated by decreasing the rate of GDP dissociation from Gas and decreasing the rate of GTP binding. Thus, BSA functions as a guanine nucleotide dissociation inhibitor for Gas . Moreover, BSA also increased the intrinsic GTPase activity of Gas , but not Gai1 , demonstrating that BSA functions as a Gas -specific GAP. Using mutants of Gas (Q227L, Q227N, R201C, and R201K), we demonstrate that BSA mediates its GAP function by modulating the ability of R201 to increase GTPase activity. Moreover, using wild-type and Q227N forms of Gas , our studies demonstrate that the GDI function of BSA decreases the ability of Gas to stimulate adenylyl cyclase. These findings assign a novel function to BSA as a regulator of G protein signaling. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: G proteins; Adenylyl cyclase; Bovine serum albumin; GTPase activity; GTPase activating protein; Guanine nucleotide dissociation inhibitor; Cyclic AMP; Stimulatory GTP binding protein of adenylyl cyclase
Heterotrimeric GTP binding proteins (G proteins1) are activated by heptahelical transmembrane receptors upon binding of their respective ligands such as hormones, neurotransmitters, and chemokines (reviewed in [1–3]). The ligand-bound, active receptors increase the exchange of GDP for GTP on the Ga subunits of the G proteins and the GTP-bound Ga subunit dissociates from the Gbc dimer. The active, GTP-bound, Ga subunits *
Corresponding author. Present address: Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA. Fax: 1-708-216-6888. E-mail address: [email protected] (T.B. Patel). 1 Abbrevations used: G protein, GTP binding protein, Gas and Gai1 , a subunits of the stimulatory and inhibitory GTP binding proteins of adenylyl cyclase, respectively; BSA, bovine serum albumin; RGS, regulators of G protein signaling; GAP, GTPase activating protein; GDI, guanine nucleotide dissociation inhibitor; DTT, dithiothreitol; GTPcS, guanosine-50 -O-(3-thiotriphosphate); C1–C2 ACV, soluble, recombinant, type V adenylyl cyclase comprising of the cytosolic domains.
subsequently regulate the downstream effectors such as adenylyl cyclase with a resultant increase (Gas ) or decrease (Gai ) in cAMP production (reviewed in [1–3]). The intrinsic GTPase activity of the Ga subunits hydrolyzes the bound GTP and the Ga subunit returns to its inactive GDP-bound state. Regulators of G protein signaling (RGS) proteins that increase the intrinsic GTPase activity of Ga subunits facilitate the inactivation of Ga subunits (reviewed in [4,5]). Therefore, the RGS proteins are also referred to as GTPase activating proteins (GAPs). To date, more than 20 RGS protein family members have been identified ([4,5] for reviews). Several of the RGS proteins are GAPs for Gi , Gz Gq , or G12=13 classes of G proteins [4,5]. However, recently, a new member of the RGS family, RGS-PX1, was also reported to act as a GAP for Gas [6]. In addition to the RGS proteins, some effectors such as PLC and adenylyl cyclase also function as GAPs for Gaq and Gas , respectively [7,8]. Bovine serum albumin (BSA), one of the most abundant proteins in the blood is synthesized and
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00263-7
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secreted by liver cells and plays a key role in the transport of fatty acids, metabolites, and drugs as well as in maintaining colloidal osmotic pressure [9,10]. Because no enzymatic or other functional activity has been identified for BSA, it has been used in numerous biochemical studies as an inert, control, protein against which active proteins are tested. Alternatively, BSA has been used to increase the protein content of samples to stabilize other proteins or protect them from proteolysis. We utilized BSA as a control protein in a screen for proteins that modulate Gas activity. Unexpectedly, we observed an inhibitory effect of BSA on the steady-state GTPase activity of Gas , without any effect on steadystate GTPase activity of Gai . These actions of BSA were obliterated by heat denaturation or trypsin digestion. Moreover, we observed that BSA acts as a specific GAP for Gas . Additionally, experiments with mutant forms of Gas showed that BSA may exert its GAP actions by modulating the ability of R201 to stabilize the oxygens on the phosphoryl (c phosphate of GTP) leaving group. Finally, we report that BSA, by regulating the activity of Gas , alters the ability of the G protein to activate adenylyl cyclase.
Materials and methods Fatty acid-free bovine albumin (fraction V; >98% pure, catalog number 152401) was purchased from ICN Biomedicals (Aurora, OH). This is the only commercially available form of albumin that is essentially free of fatty acids (<0.05 mg/g protein). Likewise, fraction V of human albumin was purchased from Sigma Chemical (St. Louis, MO; catalog number A-1653). All other chemicals were of the highest purity available from commercial sources. Recombinant Ga subunits Recombinant bovine Gas (unmodified or with Nterminus His X 6 tag) and myristoylated rat Gai1 were expressed and purified, as previously described [11–13]. The Gas used in our experiments was the short form (380 amino acid long). However, for convenience, the amino acid numbering system used is that for the long form (394 amino acids) of Gas . Mutations of Q227 to L or N, and R201 to C or K were introduced into wild type construct using mutagenic oligodeoxynucleotides and universal polymerase chain reaction. Soluble form of type V adenylyl cyclase The plasmid construct to express soluble type V adenylyl cyclase, comprising the C1 and C2 cytoplasmic domains (C1–C2 ACV) joined by an artificial linker, has
been previously described [12]. The C1–C2 ACV was expressed in TP2000 strain of Escherichia coli at 37 °C and inclusion bodies were isolated and solubilized with 6 M guanidinium hydrochloride. The denatured protein (20 lg/ml) was then refolded by dialysis (three times for 10 h each) at 4 °C against 10 volumes of buffer containing 1/10 (v/v) of PBS, pH 8.0, 10 mM sodium pyrophosphate, 100 lM ATP, 1 mM DTT, 100 lM MgCl2 , and 10% glycerol. The dialyzed protein was concentrated using Centricon Plus 80 (10,000 MW cut-off; Millipore, Bedford, MA) and buffer exchanged to 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, and 20% (v/v) glycerol. The protein at this stage was approximately 10–20% pure but demonstrated significant adenylyl cyclase activity. Steady-state GTPase activity Steady-state GTPase activity was measured in 100 ll of buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgCl2 , 1 mM DTT, 1 lM [c-32 P]GTP (5000 dpm/ pmol), and the indicated concentration of Gas or Gai1 in the presence and absence of BSA at room temperature for 10–20 min. The reactions were terminated with icecold charcoal slurry (5% (w/v) in 50 mM NaH2 PO4 ) and after centrifugation (20,000g for 10 min) aliquots of the supernatant were counted for 32 Pi release. GTPase activating protein assays The single cycle GTPase activity was measured as described by Berman et al. [14] with the following modification: Gas (2.0 lM) was first incubated at room temperature for 10 min in 50 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, and 1 lM [c-32 P]GTP (60 dpm/fmol). The temperature was lowered to 4 °C and incubation was continued for another 10 min. For time zero, 10 ll aliquots (20 pmol) were withdrawn and quenched with 0.99 ml of charcoal suspension in 50 mM NaH2 PO4 described above. Within 10 s of withdrawing the zero time point, 100 ll of the reaction mixture was diluted to 1 ml with buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgCl2 , 1 mM DTT, and 1 mM GTP. At the indicated time points, 100 ll aliquots were withdrawn and mixed with 0.9 ml of charcoal suspension. 32 Pi release was monitored as described for steady-state GTPase activity assay. GDP release and GTPcS binding GDP release was measured as follows: Gas (2.0 lM) was loaded with trace amount of [a-32 P]GTP in buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgSO4 , and 1 mM DTT for 1 h at room temperature to ensure the complete hydrolysis of bound GTP to GDP. The reaction mixture was then diluted 10 times in a buffer con-
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taining 50 mM Tris–HCl, pH 8.0, 5 mM MgSO4 , 1 mM DTT, and 1 mM GTP. Aliquots (100 ll; 20 pmol Gas ) were withdrawn at the indicated times and filtered through protran BA85 filters (Schleicher and Schuell, Keene, NH), as described previously [11]. GTPcS binding was measured by incubating Gas (200 nM) at room temperature in buffer containing 50 mM Tris–HCl, pH 8.0, 5 mM MgSO4 , 1 mM DTT, and 1 lM [35 S]GTPcS (5000 dpm/pmol). At the indicated times, 100 ll aliquots were withdrawn and filtered as described above for GDP release experiments. Adenylyl cyclase activity assays Adenylyl cylase activity was measured as described previously [12] with 40 pmol of soluble C1–C2 ACV and 41 pmol Gas in 100 ll reaction mix containing 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2 , 10 lM GTP or GTPcS, 1 mM of 3-isobutyl-1-methylxanthine, 0.1 mM ATP, 1 mM cAMP, 15,000 dpm [3 H]cAMP, and 400 dpm/ pmol [a-32 P]ATP in the presence or absence of BSA at room temperature for 20 min. The ATP regenerating system was omitted from the assay mixture so that GDP resulting from the GAP function of BSA would not be converted back to GTP. Reactions were initiated by adding the Gas and were stopped with 50 ll ice-cold, 3 stopping buffer (150 mM Tris–HCl, pH 7.4, 6% SDS, 3 mM cAMP, and 3 mM ATP). Cyclic AMP was separated by chromatography on two sequential columns of Dowex AG50 WX-4 and alumina and the amount of 32 P label in this fraction was determined by liquid scintillation counting [15].
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Results and discussion In a screen for proteins that altered Gas activity, we included BSA as a control and monitored its ability to modulate the steady-state GTPase activity of Gas . Surprisingly, BSA in a concentration-dependent manner inhibited the steady-state activity of Gas (Fig. 1A). Interestingly, BSA did not alter the steady-state GTPase activity of Gai (Fig. 1A), indicating that the effect on Gas was specific. Consistent with this latter notion, it has also been demonstrated that BSA does not have any effect on steady-state GTPase activity of transducin (Gat ) [16]. Together, these findings suggest that the inhibitory effect of BSA on steady-state GTPase activity of Gas is not due to non-specific interactions, but rather a specific interaction between Gas and BSA. BSA inhibited the steady-state GTPase activity of both N-terminal hexahistidyl-tagged or untagged Gas (data not shown), indicating that N-terminal modifications of Gas do not alter its interactions with BSA. To rule out the possibility that non-protein contaminants in the BSA may be inhibiting the steady-state GTPase activity of Gas , the experiments depicted in Fig. 1B were performed. Denaturation of BSA by heating or digestion of BSA with trypsin obliterated the ability of BSA to inhibit the steady-state GTPase activity of Gas (Fig. 1B); in the latter approach after digestion of BSA, trypsin was inactivated by heating (10 min at 100 °C). Heat inactivated trypsin, by itself, did not alter the steady-state GTPase activity of Gas (Fig. 1B). Fig. 1C shows that under the experimental conditions of Fig. 1B, trypsin almost completely digested the BSA. Note that the Coomassie
Fig. 1. Inhibition of the steady-state GTPase activity of Gas by BSA. Steady-state GTPase activities were measured for 10 min at room temperature as described in Materials and methods with either 200 nM each of Gas () or Gai1 () in presence of the indicated BSA concentrations (A). (B) Same as (A) with 200 nM of Gas except that BSA was treated as follows. BSA (300 lM) was either left untreated (BSA), or heat denatured (heated BSA) or digested with 100 lg/ml trypsin (BSA + trypsin) overnight at room temperature in buffer containing 50 mM Tris–HCl, pH 8.0, 1 mM DTT. As an additional control, 100 lg/ml trypsin without BSA (trypsin alone) was also incubated for the overnight period. Thereafter, where applicable, trypsin was inactivated by heating (100 °C for 10 min). The control and experimental samples were then centrifuged (20,000g, 10 min) and aliquots of supernatants that would yield 30 lM equivalent final concentration of BSA or 10 lg/ml trypsin were used. The effect of human serum albumin (HSA; 30 lM) on steady-state GTPase activity is also shown. (C) BSA was treated with and without trypsin as described under (B) above and 20 lg of the protein was applied to SDS–PAGE. The Coomassie stained gel shows that trypsin nearly completely digested the BSA (lane 3). Representatives of at least two experiments performed in duplicate are shown.
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stained gel (Fig. 1C) does not show any other protein bands, indicating that as reported by the manufacturer (ICN Biomedicals), the BSA is >98% pure. Together, the findings in Figs. 1B and C demonstrate that BSA, and not some non-protein contaminant, alters the steady-state GTPase activity of Gas . Human serum albumin that shares about 80% sequence homology with BSA also inhibited the steady-state GTPase activity of Gas (Fig. 1B), demonstrating the generality of the effects among albumins. The specific inhibition of steady-state GTPase activity of Gas by BSA prompted us to perform additional experiments to understand the mechanism of inhibition and its significance on the production of cAMP. The steady-state GTPase activity of Gas is determined by the rate of GTP–GDP exchange [17]. Thus, this activity could be modulated either by the inhibition of GDP release, the rate-limiting step in the steady-state GTP hydrolysis of Gas , and/or the inhibition of GTP hydrolysis per se. Therefore, first, we investigated whether BSA altered the rate of guanine nucleotide exchange on Gas . The rate of GTP–GDP exchange on Ga subunits can be monitored by following either the binding of GTPcS or the release of bound GDP. As shown in Fig. 2A, BSA decreased the rate of GTPcS binding to Gas such that the t1=2 for maximal GTPcS binding to Gas increased from 5.3 0.8 min in the absence of BSA to 14.3 0.6 min in its presence. Likewise, BSA also decreased the rate of GDP dissociation from Gas (Fig. 2B). These findings demonstrate that BSA acts as a guanine nucleotide dissociation inhibitor (GDI) for Gas and thereby contributes to the inhibition of steadystate GTPase activity of the G protein. To determine whether BSA also altered the intrinsic GTPase activity of Gas per se, the experiments depicted in Fig. 3 were performed. To monitor GTPase activity without any interference from alterations in the rate of guanine nucleotide exchange, we measured the single
Fig. 2. BSA functions as a guanine nucleotide dissociation inhibitor (GDI) for Gas . GTPcS binding (A) and GDP release (B) in the absence () or presence () of 30 lM BSA were assayed as described in Materials and methods. Total amount of Gas was 1 lg (20.5 pmol) in 100 ll of reaction mix. From the maximal binding of GTPcS (10 pmol), it was estimated that the Gas was 50% active. Representatives of two similar experiments performed in duplicate are shown.
Fig. 3. BSA accelerates the intrinsic GTPase activity of Gas , but not of Gai1 . The single cycle GTPase activities were assayed as described in Materials and methods with Gas (20.5 pmol protein, A) or Gai1 (25 pmol protein, B) in the absence () or presence () of 30 lM BSA. Gas and Gai1 were loaded with [c-32 P]GTP at room temperature and the hydrolysis was carried out at 4 °C for Gas and at room temperature for Gai1 , respectively. Representatives of at least two similar experiments are shown.
cycle GTP hydrolysis of Gas . For this purpose, Gas was first loaded with [c-32 P]GTP in presence of EDTA to block the hydrolysis of the bound GTP [14]. After incubation, the GTP bound Gas was diluted with buffer containing 1000 times excess amount of unlabeled GTP in presence of Mg2þ to initiate the hydrolysis of the bound [c-32 P]GTP in the presence and absence of BSA. This assay measures hydrolysis of the pre-bound [c32 P]GTP without having to rely on further GTP binding. As shown in Fig. 3A, BSA markedly accelerated the single cycle GTP hydrolysis of Gas but had no effect on the single cycle GTP hydrolysis of Gai1 (Fig. 3B). It has also been reported that BSA, at the concentrations used in our assays, does not affect the single cycle GTP hydrolysis of transducin (Gat ) [16]. Thus, like the GDI effect (Figs. 1 and 2), the GTPase activating property of BSA is also specifically directed at Gas . The t1=2 for complete GTP hydrolysis by Gas in the single cycle GTPase assay is about 1 min at 0 °C in the absence of BSA (Fig. 3A), which is same as that reported by Berman et al. [14], but 5 times faster than that reported by Zheng et al. [6]. However, in the presence of BSA, all of the bound GTP was hydrolyzed before the first time point (15 s) could be withdrawn (Fig. 3A). Because of the rapid GTP hydrolysis, we were unable to accurately estimate the t1=2 of complete GTP hydrolysis in the single cycle GTPase assay in presence of BSA; a conservative estimate would suggest that the intrinsic GTP hydrolysis rate of Gas in presence of BSA is at least 10 times faster than that of Gas in absence of BSA. Overall the data in Fig. 3 demonstrate that BSA increases the intrinsic GTPase activity of Gas and in this respect acts as a GTPase activating protein (GAP). Thus, just as RGS proteins act as GAPs for Gai , Gaz; and Gaq [4,5], BSA acts as a GAP for Gas but not for Gai (Fig. 3B) or Gat [16]. It has been shown that RGS4, a GAP for Gai1 , has no effect on the intrinsic GTPase activity of Gai1 Q204L
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mutant, but stimulates the GTPase activity of its R178C mutant [14]. The cognate conserved glutamine (Q227) and arginine (R201) residues on Gas are also important in regulating its GTPase activity [18,19]. Therefore, we determined whether mutations of Q227 or R201 on Gas would modulate the GDI or GAP activities of BSA. First, we determined the steady-state GTPase activity of the constitutively active Gas mutants Q227L, Q227N, R201C, and R201K. Consistent with previous findings [18,19], these mutants have a low GTPase activity (Fig. 4A). Thus, Q227L and R201C mutants of Gas exhibited less than 5% of the wild type steady-state GTPase activity (Fig. 4). Likewise, the steady-state GTPase activity of Q227N and R201K mutants was approximately 10% and 30% that of the wild type protein, respectively (Fig. 4A). Because of the low intrinsic GTPase activity, the effects of BSA, if any, on the steady-state GTPase activities of the mutants were difficult to decipher (Fig. 4A). However, like the wild-type Gas , BSA decreased the release of GDP from all of the Gas mutants, except the R201K form ( 6 10% decrease) (Fig. 4B). The precise reason(s) for the lack of an effect of BSA on GDP release from R201K Gas is presently not apparent since the GDP release from the R201C mutant was inhibited by BSA (Fig. 4B). Overall, however, it would appear that the GDI function of BSA was not altered by mutations of R201 and Q227 to cysteine and leucine or asparagine, respectively. The GTPase activity of Q227L and R201C mutants of Gas is very low and BSA did not alter the single cycle GTP hydrolysis of these mutants (Figs. 5A and C). This is in contrast to the effects of RGS4 on the equivalent mutants of Gai [14]. In this latter study, RGS4 stimu-
Fig. 4. BSA does not modulate the steady-state GTPase activity of mutant forms of Gas , but inhibits GDP release from these mutants. (A) The steady-state GTPase activities of Gas mutants were measured with 20.5 pmol each of the proteins in 100 ll of reaction mixture in the absence (open bar) or presence (filled bar) of 30 lM BSA as described in Fig. 1 and Materials and methods. (B) GDP release was measured as described in Materials and methods. Wild-type and mutant forms of Gas (20.5 pmol each) were incubated in a final volume of 10 ll with trace amount of [a-32 P]GTP in the presence of MgSO4 at room temperature for 1 h, diluted 10 times with buffer containing 1 mM GTP with or without 30 lM BSA, and incubated further for 15 min. The amount of 32 P labeled nucleotide bound to the protein was determined by filtration. Representatives of at least two similar experiments are shown.
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Fig. 5. BSA stimulates intrinsic GTPase activity of Q227N and R201K mutants, but not the Q227L and R201C mutants, of Gas . The single cycle GTPase activities were measured as described in legend to Fig. 2 and in Materials and methods. Gas mutants (20.5 pmol protein each) were loaded with [c-32 P]GTP at room temperature and the single cycle GTP hydrolysis in 100 ll reaction mixture was monitored at room temperature. (A) Q227L, (B) Q227N, (C) R201C, and (D) R201K in the absence () or presence () of 30 lM BSA. Representatives of at least two similar experiments are shown.
lated single cycle GTPase activity of R178C Gai without altering the activity of Q204L form of Gai [14]. Interestingly, however, the rate of single cycle GTP hydrolysis of the Q227N mutant of Gas was accelerated by at least 50 times to mimic the wild type protein (c.f. Figs. 5B and 3A). Likewise, the single cycle GTPase activity of the R201K mutant of Gas was also slightly augmented in the presence of BSA (Fig. 5D). Together, these results suggest that the stimulation of single cycle GTP hydrolysis of Gas by BSA is probably mediated through alterations in conformation that permit the Q227 and R201 residues to more efficiently hydrolyze the GTP; the R201 residue in heterotrimeric G protein a subunits stabilizes the c-phosphate oxygens and the phosphoryl leaving group [20,21]. These findings are different to those reported for the effects of RGS4 on Q204L and R178C mutants of Gia1 [14] and suggest that BSA and RGS may accelerate the intrinsic GTPase activity of Ga subunits through different mechanisms. The GDI and GAP actions of BSA on Gas would ultimately reduce the amount of the G protein in its active, GTP-bound, state and should decrease the ability of Gas to stimulate cAMP production by adenylyl cyclase. To test the functional significance of BSA-mediated regulation of Gas , we assessed the ability of Gas to
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stimulate the activity of partially purified, recombinant, soluble type V adenylyl cyclase (C1–C2 ACV) in the absence or presence of BSA. We have previously shown that the soluble, recombinant, C1–C2 ACV is regulated by G proteins like its full-length counterpart [12,22]. Thus, the use of soluble C1–C2 ACV permits the evaluation of G protein activity without interference from endogenous Gas that would be present in membranes of mammalian cells. In our approach, using the wild type and Q227N mutant of Gas , we monitored adenylyl cyclase activity in the presence and absence of GTP or GTPcS. The rationale in these experiments was that any effect of BSA on GTP-stimulated AC activity could be due to GDI as well as GAP functions of BSA. However, the effects of BSA on GTPcS-stimulated activity would reflect only the GDI function of BSA since GTPcS is not hydrolyzed by the G protein. The nucleotides were added to the assay mix and reactions were initiated by addition of Gas . As shown in Fig. 6A, wild-type Gas in the presence and absence of GTP did not alter adenylyl cyclase activity. Similar observations with wild-type Gas and GTP have previously been reported [19]. Moreover, BSA did not alter the activity of adenylyl cyclase in the presence of wild-type Gas with or without GTP in the assay (Fig. 6A). However, BSA inhibited by approximately 30% the GTPcS-stimulated adenylyl cyclase activity in the presence of wild type Gas (Fig. 6A). Likewise, the ability of GTPcS to stimulate adenylyl cyclase activity in the presence of Q227N Gas was also inhibited by BSA (Figs. 6A and B). These findings show that the decreased guanine nucleotide exchange on Gas in the presence of BSA (Figs. 2 and 4B) results in diminished ability of the G protein to stimulate adenylyl cyclase. Since GTP did not significantly stimulate the activity of adenylyl cyclase in the presence of wild-type Gas , we could not investigate the GAP function of BSA
using the wild-type Gas . On the other hand, because the Q227N form of Gas activates adenylyl cyclase in the presence of GTP (Fig. 6B) and because BSA significantly stimulates the GTPase activity of this mutant (Fig. 5B), it would be predicted that the ability of Q227N Gas to stimulate adenylyl cyclase activity in the presence of GTP would be markedly inhibited. However, as shown in Fig. 6B, BSA inhibited Q227N Gas stimulated adenylyl cyclase activity in the presence of GTP to the same extent as that observed with GTPcS. The residual activity was surprisingly greater than that observed with Q227N in the absence of GTP (Fig. 6B). These data suggest that either the GAP function of BSA is inconsequential in terms of regulation of adenylyl cyclase or that the hydrolyzed GTP was still bound to the G protein and the GDP along with the bound phosphate mimicked the actions of GTP. To investigate the latter possibility, we measured the binding of 32 P label from [c-32 P]GTP to wild type and Q227N forms of Gas . The binding of the nucleotide was performed in the presence of EDTA as described for the single cycle GTPase activity assays. Binding of [c-32 P]GTP was monitored just prior (0 min) to the addition of MgSO4 with or without BSA. Five minutes after addition of MgSO4 , 32 P label bound to Gas was monitored again. The premise in these experiments was that 5 min after the addition of MgSO4 either in the presence or absence of BSA, the GTP bound to the protein should be completely hydrolyzed (see Figs. 3A and 5B). Indeed, as shown in Fig. 6C, incubation for 5 min with MgSO4 in the presence or absence of BSA decreased the amount of 32 P label associated with wild type Gas . However, the binding 32 P label associated with Q227N Gas was not altered by incubation for 5 min with MgSO4 in the presence or absence of BSA (Fig. 6C). Since the addition of MgSO4 and BSA to Q227N Gas results in a very
Fig. 6. The GDI, but not the GAP, activity of BSA inhibits Gas -stimulated adenylyl cyclase activity. Adenylyl cyclase activities were measured as described in Materials and methods with 3 lg (40 pmol) C1–C2 ACV and 2 lg each (41 pmol) of Gas (wild type or Q227N mutant) in 100 ll of reaction mix in the absence (open bars), and presence (filled bars) of 30 lM BSA. (A) Experiments with wild-type Gas . (B) Same as (A), except that the Q227N Gas was preincubated for 1 h at room temperature in the presence of Mg2þ to ensure that the bound GTP on Gas was hydrolyzed. Reactions were initiated by the addition of Gas and terminated after 20 min. Data presented are means SEM of three determinations. (C) Wild type or Q227N Gas (20.5 pmol each) was incubated for 20 min at room temperature in 10 ll buffer containing 50 mM Tris–HCl, pH 8.0, 1 mM DTT, 1 lM [c-32 P]GTP, and 5 mM EDTA. Thereafter, the reactions were diluted 10 times with buffer containing 50 mM Tris–HCl, pH 8.0, 1 mM DTT, and 5 mM MgSO4 with or without 30 lM BSA. After 5 min at room temperature, the 32 P label associated with Gas was measured by filtration as described in Materials and methods. Means SE (n ¼ 3) are presented.
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rapid hydrolysis of the bound [c-32 P]GTP (Fig. 5B), the data in Fig. 6C strongly suggest that the release of the hydrolyzed phosphate from the Q227N Gas is very slow and therefore the GDP along with the bound phosphate mimics the actions of GTP. In this context, it should be noted that the experiments in the single cycle GTPase assays are terminated with an acidic slurry of charcoal and the denaturation of the G protein permits the release of 32 Pi. Overall, therefore, measurements of the functional significance of the Gas -specific GAP function of BSA remain problematic, even with the use of Q227N mutant of the G protein and the BSA-elicited inhibition of GTP-stimulated adenylyl cyclase activity with this mutant essentially represents the GDI function of BSA. In addition to showing that BSA functions as both GDI and GAP specifically for Gas , in this communication, we also report the identification of a mutant (Q227N) of Gas that is ideally suited to monitor the GAP function of proteins at room temperature. As shown in Fig. 5, the Q227N mutant permits convenient monitoring of GAP activity of proteins at room temperature. Additionally, our data show that the Q227N mutant of Gas has a high affinity for the c-phosphate of the hydrolyzed GTP and the G protein bound GDP and phosphate can mimic the GTP-bound form of the G protein and activate adenylyl cyclase. Presently, it is unclear what the physiological role and consequences of the GDI and GAP function of BSA in vivo would be. Albumin is synthesized and secreted mainly by hepatocytes [23]. However, other cells and organs such as heart, kidney, retina, and pancreas also synthesize albumin [24,25]. Since the secretory process involves vesicular trafficking [26] and because cAMP regulates transcytosis and apical secretion [27] it is tempting to speculate that BSA, by regulating cAMP production, could modulate its own secretion. However, since albumin is contained within vesicles, Gas and albumin may not normally interact with each other. Whether albumin interacts with Gas in some pathological state when albumin is not confined within vesicles is not presently known. In this context, it should be noted that the newly discovered RGS-PX1 that acts as a Gas -GAP [6] is located in endosomes and it has been suggested that RGS-PX1, by altering Gas activity, may play a role in endocytosis or recycling of proteins [6,28]. Albumin secretion from the liver is dysregulated in some chronic conditions such as diabetes, kidney disease, and cancer [10,29,30]. Whether the changes in albumin secretion are also reflected in regulation of cAMP accumulation and downstream alterations in metabolic and other biological events associated with these disease states remains to be determined. Albumin has been reported to be taken up by cells and in some cell types this results in cellular apoptosis [31,32]. Whether the induction of apoptosis by albumin involves modulation of Gas and cAMP generation remains to be determined. The transcellular
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transport of albumin in endothelial cells is mediated by a cell surface glycoprotein (gp60) that has been shown to signal vesicular trafficking via Gai and Src kinase [33]. Thus, while gp60 may activate Gai , its binding partner albumin modulates Gas activity. At the level of adenylyl cyclase, these opposing actions would reinforce each other to decrease cAMP formation. Again, this would require that albumin under some pathological states either leaks out or is released from the vesicles. Moreover, since Gas has also been reported to activate the Src family kinase Lck [34], it is possible that by modulating Gas activity, albumin may regulate the levels of both cAMP and/or the Lck signaling networks. Our findings that BSA acts as a Gas -specific GDI and GAP certainly warrant such investigations. In sum, our findings reported here assign novel functions to BSA and also show that BSA is not necessarily the inert protein that it is assumed to be.
Acknowledgments This work was supported by Grant HL59679 from the NIH (to T.B.P.) and a postdoctoral fellowship (to Z.D.) from the American Heart Association, SE Affiliate. We thank Dr. Helen Poppleton for reading the manuscript and helpful comments. We also thank Mr. David Neblett for technical assistance with this work.
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