Search

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment. IMPORTANCE Staphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections., (Copyright © 2021 Pathania et al.)

Introduction: Rats subjected to polytrauma and hemorrhage develop a coagulopathy that is similar to acute coagulopathy of trauma in humans, and is associated with a rise in prothrombin time and a fall in clot strength. Because platelet aggregation accounts for a major proportion of clot strength, we set out to characterize the effects of polytrauma on platelet function., Methods: Sprague-Dawley rats were anesthetized with isoflurane. Polytrauma included laparotomy and damage to 10 cm of the small intestines, right and medial liver lobes, right leg skeletal muscle, femur fracture, and hemorrhage (40% of blood volume). No resuscitation was given. Blood samples were taken before and after trauma for the measurement of impedance electrode aggregometry, and intracellular levels of cyclic adenosine and guanosine monophosphate (cAMP, cGMP), inositol trisphosphate (IP3), and adenosine and guanosine triphosphates (ATP, GTP)., Results: Polytrauma significantly increased the response of collagen (24%) and thrombin (12%) to stimulate platelet aggregation. However, aggregation to adenosine diphosphate (ADP) or arachidonic acid (AA) was significantly decreased at 2 (52% and 46%, respectively) and 4 h (45% and 39%). Polytrauma and hemorrhage also led to a significant early rise in cAMP (101 ± 11 to 202 ± 29 pg/mL per 1,000 platelets), mirrored by a decrease in cGMP (7.8 ± 0.9 to 0.6 ± 0.5). In addition, there was a late fall in ATP (8.1 ± 0.7 to 2.2 ± 0.6 ng/mL per 1,000 platelets) and GTP (1.5 ± 0.2 to 0.3 ± 0.1). IP3 rose initially, and then fell back to baseline., Conclusions: Polytrauma and hemorrhage led to a deficit in the platelet aggregation response to ADP and AA after trauma, likely due to the early rise in cAMP, and a later fall in energy substrates, and may explain the decrease in clot strength and impaired hemostasis observed after severe trauma.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Phospholipase D (PLD) is one of the key enzymes to mediate a variety of cellular phenomena including endocytosis, actin rearrangement, proliferation, differentiation, and migration. Dynamin as a PLD-interacting partner is a large GTP binding protein that has been considered a mechanochemical enzyme involved in endocytosis by hydrolyzing GTP. Although both PLD and dynamin have been implicated in the regulation of actin cytoskeleton, it is not known how they have a link to regulate fibronectin (FN)-induced cell spreading. Furthermore, it is unknown whether dynamin can work as a GTP-dependent regulator through its interaction with other proteins. Here, we demonstrate that PLD can be regulated by dynamin in a GTP-dependent manner and that this is critical for FN-mediated cell spreading. First, we verified that GTP-loaded dynamin can mediate the cell spreading by FN by using dynamin's GTP binding deficient mutant (K44A). Also, we confirmed that blocking the PLD activity inhibited FN-induced cell spreading, not cell adhesion. Moreover, PLD interacted with dynamin in a GTP-dependent manner in FN signaling, and this interaction was crucial for FN-induced PLD activation and cell spreading. Also, we found that PLD mutant (R128K) that didn't have GAP activity increased the GTP-dependent interaction between PLD and dynamin; it also increased PLD activity and cell spreading. These findings suggest that the observed increase in PLD activity was through boosting the binding of PLD with dynamin and it facilitated FN-induced cell spreading. These results imply that GTP-loaded dynamin, like a small GTPase could mediate a "switch on" signaling via interaction with PLD that has a role as an effector., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,β)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Drp1 is a dynamin-like GTPase that mediates mitochondrial and peroxisomal division in a process dependent on self-assembly and coupled to GTP hydrolysis. Despite the link between Drp1 malfunction and human disease, the molecular details of its membrane activity remain poorly understood. Here we reconstituted and directly visualized Drp1 activity in giant unilamellar vesicles. We quantified the effect of lipid composition and GTP on membrane binding and remodeling activity by fluorescence confocal microscopy and flow cytometry. In contrast to other dynamin relatives, Drp1 bound to both curved and flat membranes even in the absence of nucleotides. We also found that Drp1 induced membrane tubulation that was stimulated by cardiolipin. Moreover, Drp1 promoted membrane tethering dependent on the intrinsic curvature of the membrane lipids and on GTP. Interestingly, Drp1 concentrated at membrane contact surfaces and, in the presence of GTP, formed discrete clusters on the vesicles. Our findings support a role of Drp1 not only in the formation of lipid tubes but also on the stabilization of tightly apposed membranes, which are intermediate states in the process of mitochondrial fission., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Valproic acid (VPA) is an effective antiepileptic drug that may induce progressive microvesicular steatosis. The impairment of mitochondrial function may be an important metabolic effect of VPA treatment with potential adverse consequences., Objective: To investigate the influence of VPA on the activity of GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL)., Methods: The GTP- and ATP-specific SUCL activities were measured in human fibroblasts in the reverse direction, i.e. the formation of succinyl-CoA. These were assessed at different concentrations of succinate in the presence of VPA, valproyl-CoA and zinc chloride, an established inhibitor of the enzymes. Activities were measured using an optimized HPLC procedure., Results: Valproyl-CoA (1 mM) inhibited the activity of A-SUCL and G-SUCL by 45-55% and 25-50%, respectively. VPA (1 mM) had no influence on the activity of the two enzymes., Discussion: Valproyl-CoA appears to affect the activity of SUCL, especially with the ATP-specific enzyme. Considering the key role of SUCL in the Krebs cycle, interference with its activity might impair the cellular energy status. Moreover, A-SUCL is bound to the nucleoside diphosphate kinase (NDPK), which is responsible for the mitochondrial (deoxy)nucleotide synthesis. An inhibition of A-SUCL might influence the activity of NDPK inducing an imbalance of nucleotides in the mitochondria and eventually mitochondrial DNA depletion. This may account for the potential liver failure associated with valproate therapy, reported in patients with deficiencies within the mitochondrial DNA replicase system such as polymerase gamma 1.

Both subunits of αβ-tubulin that comprise the core components of microtubules bind GTP. GTP binding to α-tubulin has a structural role, whereas β-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. γ-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to γ-tubulin, we systematically mutagenized the GTP contact residues in the yeast γ-tubulin Tub4. Tub4(GTP)-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small γ-tubulin complex. However, tub4(GTP) mutants were no longer viable, and had defects in interaction between γ-tubulin and αβ-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only γ-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to γ-tubulin enhances its interaction with αβ-tubulin similarly to GTP recruitment to β-tubulin.

FtsZ polymerizes to form a cytokinetic ring at the center of a bacterial cell, which engineers bacterial cell division. FtsZ consists of N-terminal and C-terminal core domains followed by a C-terminal spacer and a conserved C-terminal tail region. Though it has been reported that both N- and C-domains can fold independently, the assembly behaviors of the N- and C-domains are not clear. In this study, we created five truncated constructs of Bacillus subtilis FtsZ, two N-domain and three C-domain constructs, and expressed and purified them. We determined their assembly properties and their effect on the assembly of full-length FtsZ to gain insight into the mechanism of FtsZ polymerization. We found that the N-domain of B. subtilis FtsZ can polymerize on its own in a GTP-dependent manner. Further, we obtained evidence indicating that the N-domain could bind to GTP but could not hydrolyze GTP by itself. In addition, the N-domain was found to inhibit the assembly of full-length FtsZ. Interestingly, the N-domain was found to enhance the GTPase activity of full-length FtsZ. An analysis of the effects of the N- and C-domains on FtsZ assembly indicated that the assembly of FtsZ might be directional. The work has provided new insight into the assembly characteristics of FtsZ domains and the mechanism of FtsZ polymerization.

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.

The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca(2+)-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.

The molecular modes of action of antipsychotic drugs are poorly understood beyond their effects at the dopamine D2 receptor. Previous studies have placed Akt signaling downstream of D2 dopamine receptors, and recent data have suggested an association between psychotic illnesses and defective Akt signaling. To characterize the effect of antipsychotic drugs on the Akt pathway, we used the model organism C. elegans, a simple system where the Akt/forkhead box O transcription factor (FOXO) pathway has been well characterized. All major classes of antipsychotic drugs increased signaling through the insulin/Akt/FOXO pathway, whereas four other drugs that are known to affect the central nervous system did not. The antipsychotic drugs inhibited dauer formation, dauer recovery, and shortened lifespan, three biological processes affected by Akt signaling. Genetic analysis showed that AKT-1 and the insulin and insulin-like growth factor receptor, DAF-2, were required for the antipsychotic drugs to increase signaling. Serotonin synthesis was partially involved, whereas the mitogen activated protein kinase (MAPK), SEK-1 is a MAP kinase kinase (MAPKK), and calcineurin were not involved. This is the first example of a common but specific molecular effect produced by all presently known antipsychotic drugs in any biological system. Because untreated schizophrenics have been reported to have low levels of Akt signaling, increased Akt signaling might contribute to the therapeutic actions of antipsychotic drugs.

Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P(2)-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P(2) is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein.

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1(+) as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745-2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1DeltaN) or the SAM domain (pob1DeltaSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1DeltaN, and pob1DeltaSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus.

Soluble guanylate cyclase (sGC) serves as a receptor for the signaling agent nitric oxide (NO). sGC synthesis of cGMP is regulated by NO, GTP, ATP, and allosteric activators such as YC-1. The guanylate cyclase activity and adenylate cyclase activity of full-length sGC and the sGC catalytic domain constructs (alpha1(cat)beta1(cat)) are reported here. ATP is a mixed-type inhibitor of cGMP production for both sGC and alpha1(cat)beta1(cat), indicating that the C-terminus of sGC contains an allosteric nucleotide binding site. YC-1 did not activate alpha1(cat)beta1(cat) or compete with ATP inhibition of cGMP synthesis, which suggests that YC-1 and ATP bind to distinct sites. alpha1(cat)beta1(cat) and NO-stimulated sGC also synthesize cAMP, but this activity is inhibited by ATP via noncompetitive substrate inhibition and by GTP via mixed-type inhibition. Additionally, the adenylate cyclase activity of purified sGC was inhibited by PC12 lysate, suggesting that an intracellular small molecule or protein regulates this activity in vivo.

The molecular basis of microtubule lattice instability derives from the hydrolysis of GTP to GDP in the lattice-bound state of alphabeta-tubulin. While this has been appreciated for many years, there is ongoing debate over the molecular basis of this instability and the possible role of altered nucleotide occupancy in the induction of a conformational change in tubulin. The debate has organized around seemingly contradictory models. The allosteric model invokes nucleotide-dependent states of curvature in the free tubulin dimer, such that hydrolysis leads to pronounced bending and thus disruption of the lattice. The more recent lattice model describes a predominant role for the lattice in straightening free dimers that are curved regardless of their nucleotide state. In this model, lattice-bound GTP-tubulin provides the necessary force to straighten an incoming dimer. Interestingly, there is evidence for both models. The enduring nature of this debate stems from a lack of high-resolution data on the free dimer. In this study, we have prepared alphabeta-tubulin samples at high dilution and characterized the nature of nucleotide-induced conformational stability using bottom-up hydrogen/deuterium exchange mass spectrometry (H/DX-MS) coupled with isothermal urea denaturation experiments. These experiments were accompanied by molecular dynamics simulations of the free dimer. We demonstrate an intermediate state unique to GDP-tubulin, suggestive of the curved colchicine-stabilized structure at the intradimer interface but show that intradimer flexibility is an important property of the free dimer regardless of nucleotide occupancy. Our results indicate that the assembly properties of the free dimer may be better described on the basis of this flexibility. A blended model of assembly emerges in which free-dimer allosteric effects retain importance, in an assembly process dominated by lattice-induced effects.

Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran--Regulator of Chromosome Condensation 1 and Ran GTPase activating protein--are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.

The interaction between prolactin and theophylline as well as between prolactin and guanosine triphosphate during Ca2+ release from intracellular stores of estradiol-treated porcine oocytes isolated from the ovary at the stage of follicular growth were studied using fluorescent Ca(2+)-sensitive probe chlortetracycline. In the absence of estradiol, prolactin or theophylline induced Ca2+ release from intracellular stores; however, no increase in Ca2+ release was observed after their combined action. Conversely, Ca2+ release from intracellular stores increased only after the combined exposure to prolactin and theophylline in the the presence of estradiol. In the absence of estradiol, guanosine triphosphate induced calcium release alone and together with prolactin. Protein kinase C regulated Ca2+ release from intracellular stores after the combined exposure to prolactin and theophylline only in the presence of estradiol; while the activation of protein kinase C required no estradiol during the combined exposure to prolactin and guanosine triphosphate. The data obtained indicate the effect of estradiol on Ca2+ release from intracellular stores after the combined exposure to prolactin and theophylline, while no such effect was observed after the combined exposure to prolactin and guanosine triphosphate.

Hyperinsulinism-hyperammonaemia syndrome (HHS) is a rare cause of congenital hyperinsulinism, due to missense mutations in the GLUD1 gene, resulting in glutamate dehydrogenase (GDH) overactivity. The aim of this study was to document the spectrum of neurological disturbances associated with HHS and to identify possible phenotype-genotype correlations. We retrospectively analyzed the neurological outcomes of 22 consecutive patients (12 males, 10 females) aged from 18 months to 40 years and diagnosed with HHS. We analyzed demographic and clinical features and neuroradiological, biochemical, and genetic findings. Fourteen patients had childhood-onset epilepsy. Learning disability was found in 17 patients. Two patients had pyramidal involvement and one had generalized dystonia. Seizures were observed in 11 of 19 patients with documented GLUD1 mutations, and nine of these 11 patients had a mutation in the guanosine triphosphate (GTP) binding site. Our data demonstrate that neurological disorders in HHS are more frequent than previously thought and might suggest that mutations in the GTP binding site of GDH could be associated with more frequent epilepsy.

In eukaryotic cells membrane compartments are connected through cargo-selective vesicle trafficking mediating the exchange of components between different organelles. This exchange is essential to maintain their structural integrity and specific composition. A fundamental regulatory step in vesicle formation is the activation of small ARF GTPases by exchanging their bound GDP for GTP, which is a prerequisite for ARF-mediated effector recruitment. Activation of ARFs is catalyzed by the characteristic SEC7 domain of guanine nucleotide exchange factors (ARF-GEFs), which are classified according to their additional protein domains.The only group of ARF-GEFs conserved in mammals, yeast and plants are the large ARF-GEFs. This review summarizes recent findings on the function of large ARF-GEFs, and the use of the inhibitor Brefeldin A as a potent tool in understanding membrane trafficking. Furthermore we highlight common themes and apparent differences in large ARF-GEF function between eukaryotic kingdoms.

When a bacterium divides, its cell wall at the division site grows radially inward like the shutter of a camera and guillotines the cell into two halves. The wall is pulled upon from inside by a polymeric ring, which itself shrinks in radius. The ring is made of an intracellular protein FtsZ (filamenting temperature sensitive Z) and thus is called the Z ring. It is not understood how the Z ring generates the required contractile force. We propose a theoretical model and simulate it to show how the natural curvature of the FtsZ filaments and lateral attraction among them may facilitate force generation.

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alphabeta-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alphabeta-tubulin. The allosteric model posits that unpolymerized alphabeta-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alphabeta-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alphabeta-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alphabeta-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alphabeta-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior.

The GTPase dynamin is a mechanochemical enzyme involved in membrane fission, but the molecular nature of its membrane interactions and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using site-directed fluorescence labeling and several independent fluorescence spectroscopic techniques, we have developed robust assays for the detection and real-time monitoring of dynamin-membrane and dynamin-dynamin interactions. We show that dynamin interacts preferentially with highly curved, PIP2-dense membranes and inserts partially into the lipid bilayer. Our kinetic measurements further reveal that cycles of GTP binding and hydrolysis elicit major conformational rearrangements in self-assembled dynamin that favor dynamin-membrane association and dissociation, respectively. Sorting nexin 9, an abundant dynamin partner, transiently stabilizes dynamin on the membrane at the onset of stimulated GTP hydrolysis and may function to couple dynamin's mechanochemical conformational changes to membrane destabilization. Amphiphysin I has the opposite effect. Thus, dynamin's mechanochemical properties on a membrane surface are dynamically regulated by its GTPase cycle and major binding partners.

Guanine-based purines have been traditionally studied as modulators of intracellular processes, mainly G-protein activity. However, they also exert several extracellular effects not related to G proteins, including modulation of glutamatergic activity, trophic effects on neural cells, and behavioral effects. In this article, the putative roles of guanine-based purines on the nervous system are reviewed, and we propose a specific guanine-based purinergic system in addition to the well-characterized adenine-based purinergic system. Current evidence suggest that guanine-based purines modulate glutamatergic parameters, such as glutamate uptake by astrocytes and synaptic vesicles, seizures induced by glutamatergic agents, response to ischemia and excitotoxicity, and are able to affect learning, memory and anxiety. Additionally, guanine-based purines have important trophic functions affecting the development, structure, or maintenance of neural cells. Although studies addressing the mechanism of action (receptors and second messenger systems) of guanine-based purines are still insufficient, these findings point to the guanine-based purines (nucleotides and guanosine) as potential new targets for neuroprotection and neuromodulation.

Tissue transglutaminase (tTG) is a multifunctional enzyme with transglutaminase crosslinking (TGase), GTP binding, and hydrolysis activities that play a role in many different disorders. We identified, characterized, and investigated the function and stability of two alternatively spliced forms of tTG using biochemical, cellular, and molecular biological approaches. Using a human aortic vascular smooth muscle cells (VSMC) cDNA library, we identified two cDNAs encoding C-terminal truncated forms, tTG(V1) and tTG(V2). tTG(V1,2) mRNAs were synthesized by a rare splicing event using alternate splice sites within exons 12 and 13 of the tTG gene, respectively. Quantitative PCR and immunoblotting demonstrated that there was unique expression and localization of tTG(V1,2) compared with tTG in human umbilical vein endothelial cells (HUVECs), VSMC, and leukocytes. The loss of C-terminal 52 amino acid residues (AAs) in tTG(V1,2) altered GTP binding, enhanced GTP hydrolysis, rendered the variants insensitive to GTP inhibition, and resulted in <10% residual Ca(+2)-dependent TGase activity. Transfection in HEK293 demonstrated a 28- and 5-fold reduction in the expression of tTG(V1) and tTG(V2), respectively, demonstrating that the C-terminal GTP-binding domain is important in stabilizing and promoting the half-life of tTG. The altered affinity for GTP allowed tTG(V1,2) to exhibit enhanced TGase activity when there is a transient increase in Ca(+2) levels. The abundance of tTG(V1,2) and its distinct intracellular expression patterns in human vascular cells and leukocytes indicate these isoforms likely have unique physiological functions.

The acrosome reaction is a regulated Ca2+-dependent secretion event required for sperm-egg interaction. Previous studies indicate that the process requires Rab3-dependent tethering of membranes, SNARE complex assembly, and Ca2+-mediated activation of synaptotagmin. Sperm are transcriptionally and translationally inactive; hence, most studies of the exocytosis mechanism are limited to membrane-permeant reagents. The effect of proteins involved in exocytosis has been assessed only in permeabilized cells. Polyarginine peptides are a powerful tool for delivering macromolecules to cells. Most reports indicate that membrane translocation of arginine-containing proteins requires endocytosis; therefore, this strategy might not be useful in sperm. However, our results indicate that GST and Rab3A, when fused with an arginine-rich peptide, were able to translocate into sperm. Moreover, membrane-permeant Rab3A initiated exocytosis when prenylated and activated with GTP. We show here that a key event after the cytoplasmic Ca2+ increase caused by progesterone is the activation of Rab3A. When active Rab3A is introduced into sperm, Ca2+ in the extracellular medium and in the cytoplasm is dispensable. However, a Ca2+ efflux from inside the acrosome is still required to achieve exocytosis. In conclusion, arginine-containing proteins can penetrate the sperm plasma membrane and thus are valuable tools to study sperm physiology in intact cells.

Lipid droplet is a cellular organelle with a neutral lipid core surrounded by a phospholipid monolayer and coated with structural as well as functional proteins. The determination of these proteins, especially their functional regulations and dynamic movement on and off droplets, holds a key to resolving the biological functions of the cellular organelle. To address this, we carried out a comprehensive proteomic study that includes a complete proteomic, a phosphoprotein proteomic, and a comparative proteomic analysis using purified lipid droplets and mass spectrometry techniques. The complete proteome identified 125 proteins of which 70 proteins had not been identified on droplets of mammalian cells previously. In phosphoprotein proteomic analysis, 7 functional lipid droplet proteins were determined to be phosphorylated, including adipose differentiation related protein (ADRP/ADFP), two Rab proteins, and four lipid metabolism enzymes, including adipose triglyceride lipase (ATGL). To understand the dynamics of lipid droplets, GTP-dependent protein recruitment was analyzed by comparative proteomics. Arf1 and some of its coatomers, three other Arfs, several other small G-proteins including 3 Rabs, and several lipid synthetic enzymes were recruited from cytosol to purified droplets. Together, the present study suggests that lipid droplet is an active and dynamic cellular organelle that governs lipid homeostasis and intracellular trafficking through protein phosphorylation as well as GTP-regulated protein translocation.

Nucleotide-specific isoforms of the tricarboxylic acid (TCA) cycle enzyme succinyl-CoA synthetase (SCS) catalyze substrate-level synthesis of mitochondrial GTP (mtGTP) and ATP (mtATP). While mtATP yield from glucose metabolism is coupled with oxidative phosphorylation and can vary, each molecule of glucose metabolized within pancreatic beta cells produces approximately one mtGTP, making mtGTP a potentially important fuel signal. In INS-1 832/13 cells and cultured rat islets, siRNA suppression of the GTP-producing pathway (DeltaSCS-GTP) reduced glucose-stimulated insulin secretion (GSIS) by 50%, while suppression of the ATP-producing isoform (DeltaSCS-ATP) increased GSIS 2-fold. Insulin secretion correlated with increases in cytosolic calcium, but not with changes in NAD(P)H or the ATP/ADP ratio. These data suggest a role for mtGTP in controlling pancreatic GSIS through modulation of mitochondrial metabolism, possibly involving mitochondrial calcium. Furthermore, in light of its tight coupling to TCA oxidation rates, mtGTP production may serve as an important molecular signal of TCA-cycle activity.

Assembly of the mitotic spindle is a classic example of macromolecular self-organization. During spindle assembly, microtubules (MTs) accumulate around chromatin. In centrosomal spindles, centrosomes at the spindle poles are the dominating source of MT production. However, many systems assemble anastral spindles, i.e., spindles without centrosomes at the poles. How anastral spindles produce and maintain a high concentration of MTs in the absence of centrosome-catalyzed MT production is unknown. With a combined biochemistry-computer simulation approach, we show that the concerted activity of three components can efficiently concentrate microtubules (MTs) at chromatin: (1) an external stimulus in form of a RanGTP gradient centered on chromatin, (2) a feed-back loop where MTs induce production of new MTs, and (3) continuous re-organization of MT structures by dynamic instability. The mechanism proposed here can generate and maintain a dissipative MT super-structure within a RanGTP gradient.

The main function of the prokaryotic translation elongation factor Tu (EF-Tu) and its eukaryotic counterpart eEF1A is to deliver aminoacyl-tRNA to the A-site on the ribosome. In addition to this primary function, it has been reported that EF-Tu from various sources has chaperone activity. At present, little information is available about the chaperone activity of mitochondrial EF-Tu. In the present study, we have examined the chaperone function of mammalian mitochondrial EF-Tu (EF-Tumt). We demonstrate that recombinant EF-Tumt prevents thermal aggregation of proteins and enhances protein refolding in vitro and that this EF-Tumt chaperone activity proceeds in a GTP-independent manner. We also demonstrate that, under heat stress, the newly synthesized peptides from the mitochondrial ribosome specifically co-immunoprecipitate with EF-Tumt and are destabilized in EF-Tumt-overexpressing cells. We show that most of the EF-Tumt localizes on the mitochondrial inner membrane where most mitochondrial ribosomes are found. We discuss the possible role of EF-Tumt chaperone activity in protein quality control in mitochondria, with regard to the recently reported in vivo chaperone function of eEF1A.

Mammalian Rho family GTPases are intracellular signal transducers known to regulate multiple signaling pathways involved in actin organization and cell proliferation. However, previous knowledge of their cellular functions came mostly from studies using a dominant-negative or constitutively active mutant expression approach in various clonal cell lines. Such an approach has increasingly been recognized to impose experimental limitations related to specificity, dosage and/or clonal variation. Recent progress in mammalian Rho GTPase cell biology by gene targeting individual Rho GTPases in mice has provided more convincing evidence of their physiological roles and signaling pathways in diverse primary cells. Although adaptive compensation by related Rho GTPase members remains a potential concern in the gene targeting approach, in many cases these studies enable an elucidation of the unique functions of individual Rho GTPases in different cell types in vivo.

Mitochondria are the major site of cellular iron utilization for the synthesis of essential cofactors such as iron-sulfur clusters and haem. In the present study, we provide evidence that GTP in the mitochondrial matrix is involved in organellar iron homoeostasis. A mutant of yeast Saccharomyces cerevisiae lacking the mitochondrial GTP/GDP carrier protein (Ggc1p) exhibits decreased levels of matrix GTP and increased levels of matrix GDP [Vozza, Blanco, Palmieri and Palmieri (2004) J. Biol. Chem. 279, 20850-20857]. This mutant (previously called yhm1) also manifests high cellular iron uptake and tremendous iron accumulation within mitochondria [Lesuisse, Lyver, Knight and Dancis (2004) Biochem. J. 378, 599-607]. The reason for these two very different phenotypic defects of the same yeast mutant has so far remained elusive. We show that in vivo targeting of a human nucleoside diphosphate kinase (Nm23-H4), which converts ATP into GTP, to the matrix of ggc1 mutants restores normal iron regulation. Thus the role of Ggc1p in iron metabolism is mediated by effects on GTP/GDP levels in the mitochondrial matrix.

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.

Defect of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT), results in Lesch-Nyhan disease (LND). It is unknown how the metabolic defect translates into the severe neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in GTP, UTP and CTP concentrations in HPRT-deficient cells. Moreover, GTP, ITP, XTP, UTP and CTP differentially support Gs-protein-mediated adenylyl cyclase (AC) activation. Based on these findings we hypothesized that abnormal AC regulation may constitute the missing link between HPRT deficiency and the neuropsychiatric symptoms in LND. To test this hypothesis, we studied AC activity in membranes from primary human skin and immortalized mouse skin fibroblasts, mouse Neuro-2a neuroblastoma cells and rat B103 neuroblastoma cells. In B103 control membranes, GTP, ITP, XTP and UTP exhibited profound stimulatory effects on basal AC activity that approached the effects of hydrolysis-resistant nucleotide analogs. In HPRT- membranes, the stimulatory effects of GTP, ITP, XTP and UTP were strongly reduced. Similarly, in human and mouse skin fibroblast membranes we also observed a decrease in GTP-stimulated AC activity in HPRT-deficient cells compared with the respective controls. In mouse Neuro-2a neuroblastoma membranes, AC activity in the presence of GTP was below the detection limit of the assay. We discuss several possibilities to explain the abnormalities in AC regulation in HPRT deficiency that encompass various species and cell types.

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia(N)) and its release by Rho*GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia(N) construct and DAD by X-ray crystallography. The core DAD region is an alpha-helical peptide, which binds in the most highly conserved region of mDia(N) using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho*GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho*GTP accelerates the dissociation of DAD from the mDia(N)*DAD complex.

How proteins are targeted and translocated mediated by signal recognition particle (SRP) in eukaryotic cell is very clear and uniform. In contrast, SRP systems are different in various kinds of prokaryotic cells, So it is difficult to identify. Nowadays, the studies of prokaryotic SRP system focus on the structure and function of Ffh, FtsY, 4.5S RNA, and GTP as a regulating molecular. Here, a description was given on research progress of constitutes, structures and functions of bacterial SRP complex proteins. The research status of streptomyces SRP pathway was also reviewed, and this study in streptomyces will be helpful to explain the molecular mechanism of prokaryotic SRP system.

This Teaching Resource provides lecture notes and slides for a class covering the structure and function of G protein-coupled receptors and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the crystal structure of rhodopsin and G protein subunits and then proceeds to describe the molecular mechanisms of receptor activation and the subsequent release of G proteins.

This Teaching Resource provides the overview to the course "Cell Signaling Systems: A Course for Graduate Students" and lays out the general principles that can be deduced from the current understanding of the organization of cell signaling pathways and networks and how information flows through these pathways and networks. In addition, the lecture provides an overview of G protein-coupled receptor (GPCR) signaling. A description of the lecture, along with a set of slides (http://stke.sciencemag.org/cgi/content/full/sigtrans;2005/270/tr4/DC1) used to present this information, is provided.

Proteins are directed to cellular compartments by specific localization signals. A GTP-driven cycle has now been identified as a mechanism for protein targeting to the nucleolus. The involvement of a GTP switch suggests that nucleolar localization can be regulated and may be responsive to extracellular stimuli via signaling pathways. The uncovered mechanism also implies that localization is determined by increased retention rather than directed targeting.

Nucleostemin (NS) was identified as a stem cell- and cancer cell-enriched nucleolar protein that controls the proliferation of these cells. Here, we report the mechanism that regulates its dynamic shuttling between the nucleolus and nucleoplasm. The nucleolar residence of nucleostemin involves a transient and a long-term binding by the basic and GTP-binding domains, and a dissociation mechanism mediated by the COOH-terminal region. This cycle is propelled by the GTP binding state of nucleostemin. We propose that a rapid nucleostemin cycle is designed to translate extra- and intra-cellular signals into the amount of nucleostemin in the nucleolus in a bidirectional and fast manner.

Microtubules are very dynamic polymers whose assembly and disassembly is determined by whether their heterodimeric tubulin subunits are in a straight or curved conformation. Curvature is introduced by bending at the interfaces between monomers. Assembly and disassembly are primarily controlled by the hydrolysis of guanosine triphosphate (GTP) in a site that is completed by the association of two heterodimers. However, a multitude of associated proteins are able to fine-tune these dynamics so that microtubules are assembled and disassembled where and when they are required by the cell. We review the recent progress that has been made in obtaining a glimpse of the structural interactions involved.

Social behaviors of most mammals are affected by chemical signals, pheromones, exchanged between conspecifics. Previous experiments have shown that behavioral responses to the same pheromone differ depending on the sex and endocrine status of the respondent. Although the exact mechanism of this dimorphism is not known, one possible contributor may be due to sexually dimorphic receptors or due to differences in central processing within the brain. In order to investigate the differences in response between male and female mice to the same pheromonal stimulus two urinary compounds (2-heptanone and 2,5-dimethylpyrazine) were used to stimulate the production of Inositol (1,4,5)-trisphosphate (IP(3)) in microvillar membrane preparations of the vomeronasal organ as an indirect measurement of pheromonal stimulation. Incubation of such membranes from prepubertal mice with urine from the same sex or opposite sex, results in an increase in production of IP(3). This stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore we found that 2-heptanone present in both male and female urine was capable of stimulating increased production of IP(3) in the female VNO but not the male VNO. Finally, 2,5-dimethylpyrazine present only in female urine was also only capable of stimulating increased production of IP(3) in the female VNO.