Guanosine Diphosphate Release Research Articles

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the β2-adrenergic receptor (β2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gαs from β2AR and Gβγ, as opposed to a slow closing of the Gαs α-helical domain (AHD). Yeast-two-hybrid screening using Gαs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gαs AHD.

Structural insights into G protein activation by D1 dopamine receptor.

G protein–coupled receptors (GPCRs) comprise the largest family of membrane receptors and are the most important drug targets. An agonist-bound GPCR engages heterotrimeric G proteins and triggers the exchange of guanosine diphosphate (GDP) with guanosine triphosphate (GTP) to promote G protein activation. A complete understanding of molecular mechanisms of G protein activation has been hindered by a lack of structural information of GPCR–G protein complex in nucleotide-bound states. Here, we report the cryo-EM structures of the D1 dopamine receptor and mini-Gs complex in the nucleotide-free and nucleotide-bound states. These structures reveal major conformational changes in Gα such as structural rearrangements of the carboxyl- and amino-terminal α helices that account for the release of GDP and the GTP-dependent dissociation of Gα from Gβγ subunits. As validated by biochemical and cellular signaling studies, our structures shed light into the molecular basis of the entire signaling events of GPCR-mediated G protein activation.

Role of ARHGEF3 as a GEF and mTORC2 Regulator.

Guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating the release of guanosine diphosphate to permit the binding of guanosine triphosphate. ARHGEF3 or XPLN (exchange factor found in platelets, leukemic, and neuronal tissues) is a selective guanine nucleotide exchange factor for Rho GTPases (RhoGEFs) that activates RhoA and RhoB but not RhoC, RhoG, Rac1, or Cdc42. ARHGEF3 contains the diffuse B-cell lymphoma homology and pleckstrin homology domains but lacks similarity with other known functional domains. ARHGEF3 also binds the mammalian target of rapamycin complex 2 (mTORC2) and subsequently inhibits mTORC2 and Akt. In vivo investigation has also indicated the communication between ARHGEF3 and autophagy-related muscle pathologies. Moreover, studies on genetic variation in ARHGEF3 and genome-wide association studies have predicted exciting novel roles of ARHGEF3 in controlling bone mineral density, platelet formation and differentiation, and Hirschsprung disease. In conclusion, we hypothesized that additional biochemical and functional studies are required to elucidate the detailed mechanism of ARHGEF3-related pathologies and therapeutics.

Conformational switch that induces GDP release from Gi

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gβγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.

Cloning of Novel Leukocytic X5 ARHGEF18 Proteoform for Functional Characterization

Rho GTPases are bio‐molecular switches in cells with activation depending on guanine nucleotide exchange factors (ARHGEFs) that facilitate GDP (guanosine diphosphate) release so GTP (guanosine triphosphate) can bind to the GTPase. When GTP is bound, GTPases are activated and contribute to cell signaling which includes regulating diverse responses such as gene transcription, cytoskeletal rearrangements, cell growth and motility. Eosinophils undergo a rapid and profound morphological change after cytokine stimulation. ARHGEF18 is abundant in eosinophils, and based on its activity in other cell types could be responsible for the formation of actin stress fibers allowing for the cell's dramatic polarization. However, our data indicates that eosinophil ARHGEF18 has an N‐terminal extension of 349 residues (part of LOC100996504 (LOC1009), which is annotated in UniProt as a unique protein) to comprise a 1364‐residue protein that has never been characterized. This project encompasses the cloning, expression, purification, and characterization of LOC1009‐ARHGEF18 (LOCGEF) and other constructs as a precursor to assessing the functional significance of LOCGEF for cytoskeleton rearrangement. The sequence of interest was created using PCR amplification of the complementary DNA sequences created from the eosinophil cellular mRNA with reverse transcription. Four separate constructs were created, one containing the entire sequence (LOCGEF), canonical ARHGEF18 without the LOC1009 sequence (p114; 1015 residues), and two sequences, one composed of LOC1009 exons 1–10 (LOC ex10; 322 residues), the other LOC1009 exons 1–11 (LOC ex11; 346 residues). The PCR product was restricted and ligated into a bacterial plasmid with a histidine tag encoded at the N‐terminus. E. coli were transformed with these plasmids using the heat shock method and plated onto kanamycin containing media. Those with the ability to grow on the media contained plasmids with the construct sequences. Bacterial expression of LOC ex10 and LOC ex11 was induced with Isopropyl β‐D‐1‐thiogalactopyranoside and the resulting proteins purified using a Ni‐NTA column. Purified LOC ex10 protein was tested in ELISA and detected using rabbit antibodies raised against the LOC1009 portion of the construct (anti‐LOC100996504 IgG; Abcam 185114). LOC ex11 has yet to be tested in ELISA. Plasmids for p114 and LOCGEF also have been created but protein yield for p114 and LOCGEF was poor in the E. coli expression system. The next step is to express these proteins in a eukaryotic system. The characterization of LOCGEF is important in understanding eosinophil cytoskeletal rearrangement needed for immune response. The function of the N‐terminal extension of LOCGEF and how it correlates with functions of the shorter isoform of ARHGEF18 previously studied in other cell types are unknown. The creation of these proteins and constructs will enable structural characterization, antibody binding, and discovery of binding partners in pull‐down assays.Support or Funding InformationNIH P01 HL088594 (Role of Eosinophils in Airway Inflammation and Remodeling)1R01AI125390‐1 (Proteomics of Eosinophil activation)

A Conserved Phenylalanine as a Relay between the α5 Helix and the GDP Binding Region of Heterotrimeric Gi Protein α Subunit

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.

Role of Small GTPase Protein Rac1 in Cardiovascular Diseases

A pathway-based genome-wide association analysis has recently identified Rac1 as one of the biologically important gene in coronary heart diseases. The role of the small GTPase Rac1 in cardiac hypertrophy and atherosclerosis has also been documented in clinical studies with the HMG-CoA reductase inhibitors and in in vitro and in vivo settings using transgenic and knockout mice. Thus, Rac1 has emerged as a new pharmacological target for the treatment of cardiovascular diseases. The activation state of Rac1 depends on the release of guanosine diphosphate and the binding of guanosine triphosphate. This cycling is regulated by the guanine nucleotide exchange factors, as activators, and by the GTPase-activating proteins. Three categories of selective Rac1 inhibitors have been developed affecting different steps of this pathway: antagonists of Rac1-guanine nucleotide exchange factor interaction, allosteric inhibitors of nucleotide binding to Rac1, and antagonists of Rac1-mediated NADPH oxidase activity. These chemical compounds have shown to selectively inhibit Rac1 activation in cultured cell lines without affecting the homologous proteins RhoA and Cdc42. Moreover, pioneer studies have been conducted with Rac1 inhibitors in in vivo experimental models of cardiovascular diseases with encouraging results. The present review summarizes the current knowledge of the role of Rac1 in cardiovascular diseases and the pharmacological approaches that have been developed to selectively inhibit its function.

Localized Deactivation Controls Net GTPase Activity

Small guanosine triphosphatases (GTPases) are switch proteins that are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). GTPases have an intrinsic GTPase activity that is stimulated by GTPase-activating proteins (GAPs), which limit the activation of their target GTPases. The GTP-bound state of GTPases is promoted by guanosine exchange factors (GEFs) that stimulate the release of GDP in exchange for GTP. Cells typically express multiple GTPases and GEFs and GAPs. Ohba et al. used engineered GTPases that produce a fluorescence resonance energy transfer (FRET) signal when active in combination with conditions that stimulate particular GEFs to show that the differential activation of GTPases at the plasma membrane or internal membranes (predominantly the perinuclear region) could not be explained by compartmentalized delivery or activation of GEFs. That is, localized activation of the GTPases persisted regardless of what GEF was used to stimulate the GTPase, including GEFs activated by soluble signals, such as adenosine 3′,5′-monophosphate (cAMP). When GTPases were mutated so that GAP interactions were blocked, they showed a more diffuse pattern of activation compared with GTPases with normal GAP interaction domains. Spatiotemporal analysis and calculation of the GEF and GAP rate constants suggested that differences in GAP activity in different regions of the cell could be responsible for the localized activation of GTPases. The data from the spatio-temporal analysis was then used to create a cellular simulation for activation of GTPase at the plasma membrane or the internal membranes that reproduced the results seen in the cells. Thus, differential inactivation of the GTPases may allow a uniformly produced activation signal to be restricted to particular subcellular locations. Y. Ohba, K. Kurokawa, M. Matsuda, Mechanism of the spatio-temporal regulation of Ras and Rap1. EMBO J. 22 , 859-869 (2003). [Abstract] [Full Text]

Localized Deactivation Controls Net GTPase Activity

Small guanosine triphosphatases (GTPases) are switch proteins that are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). GTPases have an intrinsic GTPase activity that is stimulated by GTPase-activating proteins (GAPs), which limit the activation of their target GTPases. The GTP-bound state of GTPases is promoted by guanosine exchange factors (GEFs) that stimulate the release of GDP in exchange for GTP. Cells typically express multiple GTPases and GEFs and GAPs. Ohba et al. used engineered GTPases that produce a fluorescence resonance energy transfer (FRET) signal when active in combination with conditions that stimulate particular GEFs to show that the differential activation of GTPases at the plasma membrane or internal membranes (predominantly the perinuclear region) could not be explained by compartmentalized delivery or activation of GEFs. That is, localized activation of the GTPases persisted regardless of what GEF was used to stimulate the GTPase, including GEFs activated by soluble signals, such as adenosine 3′,5′-monophosphate (cAMP). When GTPases were mutated so that GAP interactions were blocked, they showed a more diffuse pattern of activation compared with GTPases with normal GAP interaction domains. Spatiotemporal analysis and calculation of the GEF and GAP rate constants suggested that differences in GAP activity in different regions of the cell could be responsible for the localized activation of GTPases. The data from the spatio-temporal analysis was then used to create a cellular simulation for activation of GTPase at the plasma membrane or the internal membranes that reproduced the results seen in the cells. Thus, differential inactivation of the GTPases may allow a uniformly produced activation signal to be restricted to particular subcellular locations.Y. Ohba, K. Kurokawa, M. Matsuda, Mechanism of the spatio-temporal regulation of Ras and Rap1. EMBO J. 22, 859-869 (2003). [Abstract] [Full Text]

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes.

Plasma membranes (1-2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 microM [alpha-32P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37 degrees C for 1 h; during this incubation, the [32P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [32P] guanosine diphosphate (GDP). [32P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca2+, Mg2+, Mn2+, and Zn2+ maximally inhibited GDP release by 80-90%, whereas, 5 mM Cu2+ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One microM Gpp(NH)p (5'-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [32P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1-100 nM glucagon (used as a positive control) stimulated [32P]GDP release by about 17% (P < .05); similarly, 0.1-100 nM insulin stimulated [32P]GDP release by 10-13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [32P]GDP release by 7-10%. In the rat membranes, 10 nM insulin stimulated [32P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [32P]GDP release by about 9% at 2 and 4 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca 2+ potentiates corticotropin-induced, but not isoproterenol-induced, [ 3H]guanosine diphosphate release in rat adipocyte membranes

EGTA abolished corticotropin (ACTH)-stimulated adenylate cyclase activity in rat adipocyte membranes. In contrast, the potency of guanosine triphosphate (GTP) stimulation of adenylate cyclase activated with ACTH was greater in the presence of Ca 2+ (1 mmol/L). EGTA (1 mmol/L) powerfully inhibited ACTH-stimulated [ 3H]guanosine diphosphate (GDP) release from membranes prelabeled with [ 3H]GTP in the presence of isoproterenol (ISO) or ACTH, whereas Ca 2+ significantly increased it. In contrast, neither EGTA nor Ca 2+ affected ISO-stimulated [ 3H]GDP release. These data clearly show that Ca 2+ is necessary for the binding of ACTH to its receptor, and that Ca 2+ stimulates the interaction of the ACTH-occupied receptor with GTP-binding proteins.

Enhancement of the GDP-GTP Exchange of RAS Proteins by the Carboxyl-Terminal Domain of SCD25

In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP). In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase. The 3'-terminal part of SCD25, a gene of S. cerevisiae structurally related to CDC25, can suppress the requirement for CDC25. Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP. This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein. Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.