Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.     

Structure-function relationships of elongation factor Tu as studied by mutagenesis.

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity

Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.     

Crystal structure of transglutaminase 3 in complex with GMP: structural basis for nucleotide specificity

Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.     

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.     

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.     

Loss of the effector function in a transducin-alpha mutant associated with Nougaret night blindness

A missense mutation, G38D, was found in the rod transducin alpha subunit (Galpha(t)) in individuals with the Nougaret form of dominant stationary night blindness. To elucidate the mechanism of Nougaret night blindness, we have examined the key functional properties of the mutant transducin. Our data show that the G38D mutation does not alter the interaction between Galpha(t) and Gbetagamma(t) or activation of transducin by photoexcited rhodopsin (R*). The mutant Galpha(t) has only a modestly (approximately 2.5-fold) reduced k(cat) value for GTP hydrolysis. The GTPase activity of Galpha(t)G38D can be accelerated by photoreceptor regulator of G protein signaling, RGS9. Analysis of the Galpha(t)G38D interaction with cGMP phosphodiesterase revealed marked impairment of the mutant effector function. Galpha(t)G38D completely fails to bind the inhibitory PDE gamma subunit and activate the enzyme. Altogether, our results demonstrate a novel molecular mechanism in dominant stationary night blindness. In contrast to known forms of the disease caused by constitutive activation of the visual cascade, the Nougaret form has its origin in attenuated visual signaling due to loss of effector function by transducin G38D mutant.     

rasH mutants deficient in GTP binding.

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.     

Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function

The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector-binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant-GDP and mant-GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg-like protein results in the lack of function in vivo.     

Structural basis for the coordinated regulation of transglutaminase 3 by guanine nucleotides and calcium/magnesium

Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.     

The GTP-binding domain of McrB: more than just a variation on a common theme?

The methylation-dependent restriction endonuclease McrBC from Escherichia coli K12 cleaves DNA containing two R(m)C dinucleotides separated by about 40 to 2000 base-pairs. McrBC is unique in that cleavage is totally dependent on GTP hydrolysis. McrB is the GTP binding and hydrolyzing subunit, whereas MrC stimulates its GTP hydrolysis. The C-terminal part of McrB contains the sequences characteristic for GTP-binding proteins, consisting of the GxxxxGK(S/T) motif (position 201-208), followed by the DxxG motif (position 300-303). The third motif (NKxD) is present only in a non-canonical form (NTAD 333-336). Here we report a mutational analysis of the putative GTP-binding domain of McrB. Amino acid substitutions were initially performed in the three proposed GTP-binding motifs. Whereas substitutions in motif 1 (P203V) and 2 (D300N) show the expected, albeit modest effects, mutation in the motif 3 is at variance with the expectations. Unlike the corresponding EF-Tu and ras -p21 variants, the D336N mutation in McrB does not change the nucleotide specificity from GTP to XTP, but results in a lack of GTPase stimulation by McrC. The finding that McrB is not a typical G protein motivated us to perform a search for similar sequences in DNA databases. Eight microbial sequences were found, mainly from unfinished sequencing projects, with highly conserved sequence blocks within a presumptive GTP-binding domain. From the five sequences showing the highest homology, 17 invariant charged or polar residues outside the classical three GTP-binding motifs were identified and subsequently exchanged to alanine. Several mutations specifically affect GTP affinity and/or GTPase activity. Our data allow us to conclude that McrB is not a typical member of the superfamily of GTP-binding proteins, but defines a new subfamily within the superfamily of GTP-binding proteins, together with similar prokaryotic proteins of as yet unidentified function.     

The GTP-binding protein of rod outer segments. I. Role of each subunit in the GTP hydrolytic cycle

The GTP-binding protein of Bufo marinus rod outer segments (ROS) is composed of 3 subunits: G alpha, 39,000; G beta, 36,000; and G gamma, approximately 6,500. A stepwise analysis of the GTP hydrolytic cycle (GTP binding, GTP hydrolysis, and GDP release) was facilitated by using purified subunits of the GTP-binding protein. When G alpha and G beta, gamma concentrations were held constant, the initial rate of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha was dependent upon the amount of bleached rhodopsin present (as illuminated, urea-washed ROS disc membranes). When G alpha and the quantity of these membranes was held constant, the initial rate of GTP gamma-s binding to G alpha was markedly enhanced by increasing the amount of G beta, gamma. G beta preparations (free of G gamma) also stimulated the binding of GTP gamma-s to G alpha to the same extent as G beta, gamma preparations, suggesting that G gamma is not an essential component of the G beta, gamma-dependent stimulation of the rate of GTP gamma-s binding to G alpha. Nonlinear regression analysis revealed a single class of binding sites with an apparent stoichiometry of 1 mol of site/mol of G alpha under optimal binding conditions. Following GTP binding to G alpha, the GTP X G alpha complex dissociates from G beta, gamma which remains primarily bound to the ROS disc membranes. Moreover, while GTP remains in excess, the rates of GTP hydrolysis exhibited saturation in the presence of increasing amounts of G beta, gamma. Nonlinear regression analysis of these data argues against a direct role for G beta, gamma in the hydrolysis of GTP. Thus, both topologic and kinetic data support the concept that GTP hydrolysis is carried out by G alpha alone. After hydrolysis of GTP, the GDP X G alpha complex returned to the ROS disc membrane when G beta, gamma was present on the membrane surface, in the presence and absence of light. Without guanine nucleotides GDP release occurred in the presence of illuminated ROS disc membranes and G beta, gamma. Guanine nucleotides (GTP gamma-s approximately equal to GTP approximately equal to guanosine 5'-(beta, gamma-imido)triphosphate greater than GDP) could effectively displace GDP from G alpha under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic analysis of the interaction of tissue transglutaminase with a nonpeptidic slow-binding inhibitor

Tissue transglutaminase (TGase) is a Ca2+-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues and is allosterically regulated by GTP. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate as part of these disorders. Given the role that TGase plays in neurodegenerative disorders, we initiated a research program to discover inhibitors of this enzyme that might ultimately be developed into therapeutic agents. To identify such inhibitors, we screened 110,000 druglike compounds for their ability to inhibit TGase [Case, A., et al. (2005) Anal. Biochem. 338, 237-244]. In this paper, we report the kinetics of interaction of human TGase with one of the inhibitors that we identified, LDN-27219. We found that this compound is a reversible, slow-binding inhibitor that appears not to bind at the enzyme's active site but rather at the enzyme's GTP site, or a site that regulates binding of GTP. Interestingly, the potency and kinetics of inhibition are dependent on substrate structure and suggest a novel mechanism of inhibition that involves differential binding of LDN-27219 to multiple conformational states of this enzyme.     

Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.     

Coordinate regulation of G protein signaling via dynamic interactions of receptor and GAP

Signal output from receptor-G-protein-effector modules is a dynamic function of the nucleotide exchange activity of the receptor, the GTPase-accelerating activity of GTPase-activating proteins (GAPs), and their interactions. GAPs may inhibit steady-state signaling but may also accelerate deactivation upon removal of stimulus without significantly inhibiting output when the receptor is active. Further, some effectors (e.g., phospholipase C-beta) are themselves GAPs, and it is unclear how such effectors can be stimulated by G proteins at the same time as they accelerate G protein deactivation. The multiple combinations of protein-protein associations and interacting regulatory effects that allow such complex behaviors in this system do not permit the usual simplifying assumptions of traditional enzyme kinetics and are uniquely subject to systems-level analysis. We developed a kinetic model for G protein signaling that permits analysis of both interactive and independent G protein binding and regulation by receptor and GAP. We evaluated parameters of the model (all forward and reverse rate constants) by global least-squares fitting to a diverse set of steady-state GTPase measurements in an m1 muscarinic receptor-G(q)-phospholipase C-beta1 module in which GTPase activities were varied by approximately 10(4)-fold. We provide multiple tests to validate the fitted parameter set, which is consistent with results from the few previous pre-steady-state kinetic measurements. Results indicate that (1) GAP potentiates the GDP/GTP exchange activity of the receptor, an activity never before reported; (2) exchange activity of the receptor is biased toward replacement of GDP by GTP; (3) receptor and GAP bind G protein with negative cooperativity when G protein is bound to either GTP or GDP, promoting rapid GAP binding and dissociation; (4) GAP indirectly stabilizes the continuous binding of receptor to G protein during steady-state GTPase hydrolysis, thus further enhancing receptor activity; and (5) receptor accelerates GDP/GTP exchange primarily by opening an otherwise closed nucleotide binding site on the G protein but has minimal effect on affinity (K(assoc) = k(assoc)/k(dissoc)) of G protein for nucleotide. Model-based simulation explains how GAP activity can accelerate deactivation >10-fold upon removal of agonist but still allow high signal output while the receptor is active. Analysis of GTPase flux through distinct reaction pathways and consequent accumulation of specific GTPase cycle intermediates indicate that, in the presence of a GAP, the receptor remains bound to G protein throughout the GTPase cycle and that GAP binds primarily during the GTP-bound phase. The analysis explains these behaviors and relates them to the specific regulatory phenomena described above. The work also demonstrates the applicability of appropriately data-constrained system-level analysis to signaling networks of this scale.     

Activation of the Ras-ERK pathway inhibits retinoic acid-induced stimulation of tissue transglutaminase expression in NIH3T3 cells

Retinoic acid (RA) is a potent activator of tissue transglutaminase (TGase) expression, and it was recently shown that phosphoinositide 3-kinase (PI3K) activity was required for RA to increase TGase protein levels. To better understand how RA-mediated TGase expression is regulated, we considered whether co-stimulation of NIH3T3 cells with RA and epidermal growth factor (EGF), a known activator of PI3K, would facilitate the induction or increase the levels of TGase expression. Instead of enhancing these parameters, EGF inhibited RA-induced TGase expression. Activation of the Ras-ERK pathway by EGF was sufficient to elicit this effect, since continuous Ras signaling mimicked the actions of EGF and inhibited RA-induced TGase expression, whereas blocking ERK activity in these same cells restored the ability of RA to up-regulate TGase expression. However, TGase activity is not antagonistic to EGF signaling. The mitogenic and anti-apoptotic effects of EGF were not compromised by TGase overexpression, and in fact, exogenous TGase expression promoted basal cell growth and resistance to serum deprivation-induced apoptosis. Moreover, analysis of TGase expression and GTP binding activity in a number of cell lines revealed high basal TGase GTP binding activity in tumor cell lines U87 and MDAMB231, indicating that constitutively active TGase may be a characteristic of certain cancer cells. These findings demonstrate that TGase may serve as a survival factor and RA-induced TGase expression requires the activation of PI3K but is antagonized by the Ras-ERK pathway.     

Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity.

Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).