Binds to HUWE1 and represses its ubiquitin ligase activity. May play a role in controlling cell proliferation and apoptosis during mammary gland development.
Isoform smARF may be involved in regulation of autophagy and caspase-independent cell death; the short-lived mitochondrial isoform is stabilized by C1QBP : used in vesicular proteins trafficking;. That Rab regulates endocytic recycling. Acts as a major regulator of membrane delivery during cytokinesis.
Participates in the sorting and basolateral transport of CDH1 from the Golgi apparatus to the plasma membrane. May also play a role in melanosome transport and release from melanocytes regulate vesicular trafficking pathways. Involved in a microtubule-dependent signal that is required for the myosin contractile ring formation during cell cycle cytokinesis.
Plays an essential role in cleavage furrow formation. Required for the apical junction formation of keratinocyte cell-cell adhesion. Stimulates PKN2 kinase activity. May be an activator of PLCE1. Essential for the SPATAmediated regulation of cell migration and adhesion assembly and disassembly. In turn, membrane-bound APC allows the localization of the MACF1 to the cell membrane, which is required for microtubule capture and stabilization.
Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Recently, an NMR study did not detect dimerization using the G-domain without the anchor region and without a bilayer Kovrigina et al.
The latter can possibly promote a preorientation that leads to an entropic benefit for dimerization, similar to a crosslink Zaman et al. As H-, N- and K-Ras have differences in their helix 4 and helix 5 regions, it is possible that these lead to the variable behaviors observed for the different isoforms Parker and Mattos, Thus, the unambiguous characterization of the correct dimerization interface is still challenging. Using the ATR technique further allows us to study the role of membrane binding of Ras.
This opens the door for reconstitution experiments at the membrane in a near native but accurately defined system. Goody, Alfred Wittinghofer and Herbert Waldmann is very gratefully acknowledged. Ahearn, I. Regulating the regulator: post-translational modification of RAS.
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The Ras G domain lacks the intrinsic propensity to form dimers. Li, G. Ligeti, E. Inhibition and termination of physiological responses by GTPase activating proteins. After s, activated receptor was instantaneously removed and the system was allowed to return to the original steady-state.
To reveal the mechanisms underlying the observed dynamics, the fractional activity Z , the fluxes and the concentrations of all species were computed as a function of time. Figure 7 shows a typical simulated output pulse shape Z as function of time and the reaction pathways responsible for it. We also surveyed the response to a pulse over a grid of receptor and GAP concentrations 2, grid points. Locations where the higher GAP concentration accelerated signal at least two-fold are shown on inset of Figure 7.
The mechanisms underlying the dynamic response were studied at selected points Results. Differential equations used to model the reactions of the GTPase cycle shown in Figure 1 of the main text. Cost minimization during simulated annealing to fit parameters of the GTPase model under path-independence constraints Materials and Methods. Blue symbols denote accepted moves; others are red. The green triangle shows the best fit to the data. Initial points are off-scale. Two-stage cost minimization.
The initial stage used non-conformance to stoichiometric network theory as a penalty Materials and Methods. Strict path independence was enforced following step dashed line. The small cost offset between the two minimization methods has been removed from the left part of the graph. Blue triangles show solutions accepted by the minimization algorithm; red dots are rejected solutions. Analysis of the thermal ensemble of kinetic parameters about the best fit to data.
A Fractional projections of eigenvectors of the covariance matrix onto the rate constants of the model are shown as colors on the calibration bar. Eigenvectors are ordered left to right from large eigenvalues to small eigenvalues floppy to stiff. Eigenvectors 1 to 35 describe the degrees of freedom in the fit; the remaining 13 describe the thermodynamic constraints.
B Forward and reverse kinetic parameters for each reaction are plotted according to projections of the 48 eigenvectors on each parameter. Projections of constraint eigenvectors are shown in red to highlight the forward to reverse indeterminacy innate to a thermodynamically constrained fit. Higher values of projections indicate that parameters are more independently measurable. Higher eigenvector numbers 1 to 35 indicate greater stiffness.
GTPase cycle fluxes determine fractional activation during steady-state turnover. Lengths of the arrows denotes relative flux. Where no arrows are visible, flux is not distinguishable from zero. Note that RGAT is the central intermediate for all utilized pathways. See Figure S6 for details. Fluxes are calculated from RGT and are therefore shown with negative values.
Accumulation of individual activated G q species changes with increasing concentrations of GAP. The steady-state concentration of each activated GTP-bound species right axis and net fractional G q activation Z left axis are plotted as functions of the total concentration of GAP along the vertical line in Figure 6.
We are grateful to Lisa Kinch UTSW for first proposing and stressing the need for the thermodynamically complete model shown in Figure 1. Performed the experiments: WT. Performed computational analyses, fitting, and simulations: MT. Abstract 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.
Author Summary Throughout the eukaryotes, G proteins convey information from receptors for diverse stimuli—neurotransmitters, hormones, light, odors, and pheromones—to intracellular regulatory proteins collectively known as effectors. Introduction G protein-mediated signaling modules display a variety of dynamic input-output behaviors despite their use of a single, relatively simple biochemical mechanism. Download: PPT. Figure 1. Figure 2. Agreement of simulations blue with GTPase rate data black.
Figure 3. Rate constants for the G q -catalyzed GTPase cycle obtained by fitting steady-state kinetic data. Figure 4. Reconstruction of model parameters by fitting synthetic data. Cooperative Interactions of G Protein, Receptor, and GAP The parameter set shown in Figure 3 and Table S2 provides the first reasonably complete set of experimentally determined rate constants for a G protein signaling module, and thus provides insights into regulatory interactions that were not previously accessible.
Table 1. Effect of receptor on nucleotide exchange kinetics and equilibria. Table 2. Table 3. Effect of receptor on affinity of G q for GAP. Figure 5. Steady-state activation of G q under experimental conditions in reconstituted vesicles. Figure 6. Steady-state activation of G q under cellular conditions. Coordinate Regulation of Signal Output by Receptor and GAP To examine the overall regulatory behavior of the G protein module, we used the complete reaction model and average fitted parameter set to simulate signal output as the fraction Z of all G protein complexes to which GTP is bound.
G Protein Activation under Cellular Conditions We also used the model and parameter set to simulate G protein activation under typical cytoplasmic conditions—0. Figure 7.
GAP-promoted deactivation of G q after removal of receptor. Discussion Data-Constrained Modeling of a G Protein Signaling Module A mechanistic model of signal transduction should provide quantitative understanding of how time-dependent outputs arise from the underlying binding, conformational and chemical reactions.
Cooperative Interactions in G Protein Signaling Because many of the important rate constants that describe the G protein signaling module were reasonably well determined by the fits to experimental data, this study identified several new regulatory interactions that control the rate and extent of G protein activation.
Transient Responses and Signaling Dynamics Simulations based on the parameterized model suggest mechanisms for how GAP activity promotes fast deactivation when agonist is removed without attenuating the signal while agonist is present. Model Implementation The biochemical model is implemented as a system of 14 ordinary differential equations that describe the concentrations of each of the protein species shown in Figure 1 , plus free receptor and GAP Figure S1.
Impulse Response To simulate the response of G protein signaling to addition and removal of agonist, we first brought the system to an initial steady-state without receptor. Supporting Information. Text S1. Reaction volumes and second order rate constants. Text S2. Determining the quality of the fit using thermal ensembles. Text S3. Interactive regulation by receptor and GAP under cytosolic conditions. Table S1. GTPase assays used for fitting the model.
Table S2. Values of parameters. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Acknowledgments We are grateful to Lisa Kinch UTSW for first proposing and stressing the need for the thermodynamically complete model shown in Figure 1. References 1. Annu Rev Biochem — View Article Google Scholar 2. View Article Google Scholar 3. Annu Rev Pharmacol Toxicol — View Article Google Scholar 4. View Article Google Scholar 5.
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Mol Cell Biochem 1— Dodson and D. Trentham for critical reading of this manuscript, and A. Savoia Trieste and A. Thompson ESRF for beamline assistance. Katrin Rittinger, Philip A.
Walker, John F. Eccleston, Kurshid Nurmahomed, Steven J. You can also search for this author in PubMed Google Scholar. Correspondence to Steven J. Reprints and Permissions. Rittinger, K. Download citation. Received : 25 April Accepted : 25 June Issue Date : 14 August Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.
Nature Communications Molecular Neurobiology Tumor Biology Nature Cell Biology By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Abstract Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions 1 , 2.
You have full access to this article via your institution. Main The crystal structures of Ras and Rho proteins are similar 13 , 14 , 15 , 16 , and of the 41 common residues, most are located around the guanine-nucleotide-binding site. Full size image. Methods Crystallization.
Table 1 Table 1 Summary of crystallographic analysis Full size table. References 1 Bourne, H. Article Google Scholar 29 Kraulis, P. Article Google Scholar Download references. Acknowledgements We thank M. View author publications.
Rights and permissions Reprints and Permissions. About this article Cite this article Rittinger, K. Copy to clipboard. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines. Search Search articles by subject, keyword or author.
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