DOCSLIB.ORG

The Gtpase Activating Factor for Transducin in Rod Photoreceptors Is the Complex Between RGS9 and Type 5 G Protein ␤ Subunit

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Gtpase Activating Factor for Transducin in Rod Photoreceptors Is the Complex Between RGS9 and Type 5 G Protein ␤ Subunit Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1947–1952, March 1999 Biochemistry The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein b subunit ELINA R. MAKINO,JASON W. HANDY,TIANSEN LI, AND VADIM Y. ARSHAVSKY* Department of Ophthalmology, Harvard Medical School, and the Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Communicated by Thaddeus P. Dryja, Harvard Medical School, Boston, MA, December 28, 1998 (received for review December 14, 1998) ABSTRACT Proteins of the regulators of G protein sig- then some transducin molecules would never activate PDE and naling (RGS) family modulate the duration of intracellular signal amplification would be diminished. Therefore, making signaling by stimulating the GTPase activity of G protein a the GTPase activation contingent on transducin association subunits. It has been established that the ninth member of the with PDEg ensures both high efficiency of signal transmission RGS family (RGS9) participates in accelerating the GTPase between transducin and PDE and timely photoresponse re- activity of the photoreceptor-specific G protein, transducin. covery. This process is essential for timely inactivation of the photo- Although the general scheme for transducin GTPase regu- transduction cascade during the recovery from a photore- lation, outlined above is well supported by experimental data, sponse. Here we report that functionally active RGS9 from several fundamental mechanistic questions are not resolved. It vertebrate photoreceptors exists as a tight complex with the remains unclear why the effect of PDEg observed both with b long splice variant of the G protein subunit (Gb5L). RGS9 RGS9-containing photoreceptor membranes (7, 8) and intact and Gb5L also form a complex when coexpressed in cell photoreceptors (10) is substantially more pronounced than its culture. Our data are consistent with the recent observation effect in reconstituted system with recombinant catalytic do- that several RGS proteins, including RGS9, contain G protein main of RGS9 (9). It is also not understood why the RGS9 g-subunit like domain that can mediate their association with catalytic domain is able to support the rate of transducin Gb5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., GTPase at physiologically fast rate, but intact RGS9 in the Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. photoreceptor requires cooperation with PDEg. A major dif- & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, ficulty in addressing these questions is that functionally active 13307–13312). We report an example of such a complex whose full-length RGS9 has never been purified or expressed. One cellular localization and function are clearly defined. possibility is that RGS9 in photoreceptors exists as a multiple subunit complex and that some of its properties could be Heterotrimeric G proteins act as molecular switches that relay manifested only within such a complex. excitation from activated receptors to effector molecules, such In this study we explored the hypothesis that RGS9 in as enzymes or ion channels. A G protein becomes activated photoreceptors exists as a multiple subunit complex and found upon the receptor-stimulated binding of GTP to its a subunit that indeed it is present as a tight complex with the long splice b and continues to modulate the activity of the effector until variant of the type 5 subunit of G proteins (Gb5L) that cannot bound GTP is hydrolyzed (reviewed in refs. 1 and 2). In many be separated from RGS9 under nondenaturing conditions. The signaling pathways, the duration of the signal under physio- existence of this complex has been shown by three compli- logical conditions is much shorter than would be predicted mentary approaches. First, RGS9 and Gb5L comigrate when from the intrinsic rate of a subunits of G proteins (Ga) GTPase photoreceptor membrane proteins were subjected to various activity. This is because GTPase activities of many Gas are chromatographic procedures. Second, antibodies raised dramatically accelerated by RGS (regulators of G protein against each of these proteins quantitatively precipitated both signaling) proteins or by the G protein effectors (reviewed in RGS9 and Gb5L. Finally, these proteins were coimmunopre- refs. 3–6). The phototransduction cascade of vertebrate pho- cipitated after expression in cell culture. toreceptors represents one of the most sophisticated examples of such regulation where the GTPase activity of the G protein, transducin, is substantially enhanced by the cooperative action MATERIALS AND METHODS of RGS9 and the g subunit of the effector of transducin, cGMP Purification and Washing of Rod Outer Segment (ROS) phosphodiesterase (PDEg) (7–10). The role of RGS9 is to Membranes. ROS were purified from frozen retinas (TA & provide transducin with the RGS homology domain, which WL Lowson, Lincoln, NE) under IR illumination as described acts catalytically in stimulating the rate of transducin GTPase. (11). To obtain membranes lacking most peripheral proteins g PDE itself does not activate transducin GTPase but it en- but retaining an active RGS9–Gb5L complex, ROS were hances the catalytic action of RGS9. The degree of this washed under IR illumination twice with isotonic buffer potentiation observed in physiologically intact photoreceptors containing 100 mM KCl, 2 mM MgCl , 1 mM DTT, and 10 mM ' 2 is 7-fold (10). We believe that this ability of PDEg to TriszHCl (pH 7.5) and three times by a hypotonic buffer potentiate RGS9 action is essential for photoreceptor function. containing 0.5 mM EDTA and 5 mM TriszHCl (pH 7.5). When a rod photoreceptor is hit by photon of light it has to Urea-washed photoreceptor discs lacking the activity of RGS9 perform two tasks. First, it has to transmit the signal from were obtained as described (12). Rhodopsin concentration in excited rhodopsin to PDE with high efficiency. Second, it has to inactivate all activated proteins in the cascade, including a transducin, within a fraction of 1 s. If transducin is allowed to Abbreviations: RGS, regulators of G protein signaling; Ga, subunits of G proteins; Gb, b subunits of G proteins; Gg, g subunits of G be discharged by RGS9 before it forms a complex with PDE, proteins; ROS, rod outer segments; PDE, type 6 cGMP phosphodi- esterase from ROS; PDEg, the g subunit of PDE; GGL domain, G g b The publication costs of this article were defrayed in part by page charge protein subunit-like domain; GAP, GTPase activating protein; G 5L, long splice variant of Gb. payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed at: Howe Laboratoryy accordance with 18 U.S.C. §1734 solely to indicate this fact. Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston PNAS is available online at www.pnas.org. MA 02114. e-mail: [email protected]. 1947 Downloaded by guest on September 30, 2021 1948 Biochemistry: Makino et al. Proc. Natl. Acad. Sci. USA 96 (1999) y all membrane preparations was determined spectrophoto- for SDS PAGE. Antibodies against Gg2 were obtained from metrically (13). Santa Cruz Biotechnology. Preparation of Proteins. Transducin was purified from ROS Mass Spectrometric Fingerprinting. Mass spectrometric fin- as described (14). The only modification was that we bleached gerprinting analysis was performed by J. Lee at the Molecular rhodopsin in the retinas before ROS purification to increase Biology Core Facilities at the Dana–Farber Cancer Institute transducin yield by forming a tight complex between bleached (Boston). Protein mixtures were subjected to SDSyPAGE, gels rhodopsin and transducin, thus preventing transducin loss were stained by Coomassie blue G-250 (Sigma), and bands from ROS membranes during ROS isolation. Transducin containing proteins of interest were cut from the gel and pro- concentration was first estimated by the Bradford assay (15), teolyzed by trypsin as described (21). The molecular masses of and then the exact concentration of active transducin in each proteolytic peptide fragments were determined by MALDI-TOF preparation was determined by measuring the maximal (matrix-assisted laser desorption ionization-time of flight) spec- amount of rhodopsin-catalyzed guanosine 59[g-thio]triphos- trometry on Voyager-DE STR instrument (Perceptive Biosys- phase (GTP[gS]) binding (16). tems, Cambridge, MA). The values for molecular masses in Recombinant PDEg was purified by a combination of cation- digests were compared with the values from theoretical digests exchange and reverse-phase chromatography (17) from Esch- by using PROTEINPROSPECTOR MS-Fit software (available via erichia coli strain BL21 DE3 transformed with an expression internet at http:yyprospector.ucsf.edu). The probability of the plasmid containing a cDNA encoding PDEg (18). The PDEg positive protein identification was assessed with the molecular concentration was determined spectrophotometrically at 280 weight search score described (22). Because the amount of nm by using a molar extinction coefficient of 7,100. protein in the bands originated from the gel filtration fractions GTPase Measurements. Transducin GTPase activity was was too small to obtain reliable data, we performed the , determined by the single-turnover ([GTP] [transducin]) analysis with the Gb5L immunoreactive band obtained from the technique described in detail previously (7, 19, 20). The concentrated alkaline extract of washed ROS membranes. The measurements were conducted at room temperature (22– extraction was performed as described (23) by 100 mM z 24°C) in a buffer containing 10 mM Tris HCl (pH 7.8), 100 mM Na2CO3 with the pH adjusted to 12.0 by NaOH. Before the y NaCl, 8 mM MgCl2, and 1 mM DTT. The urea-treated SDS PAGE separation, proteins in the extract were concen- photoreceptor discs used as a source of rhodopsin were trated by precipitation with 3% trichloroacetic acid. bleached on ice immediately before the experiments. For cDNA Cloning and in Vitro Expression. A mouse retinal measuring the activity in the gel-filtration chromatography cDNA library (provided by D.
Load more

Recommended publications
  • Ras Gtpase Chemi ELISA Kit Catalog No
    Ras GTPase Chemi ELISA Kit Catalog No. 52097 (Version B3) Active Motif North America 1914 Palomar Oaks Way, Suite 150 Carlsbad, California 92008, USA Toll free: 877 222 9543 Telephone: 760 431 1263 Fax: 760 431 1351 Active Motif Europe Waterloo Atrium Drève Richelle 167 – boîte 4 BE-1410 Waterloo, Belgium UK Free Phone: 0800 169 31 47 France Free Phone: 0800 90 99 79 Germany Free Phone: 0800 181 99 10 Telephone: +32 (0)2 653 0001 Fax: +32 (0)2 653 0050 Active Motif Japan Azuma Bldg, 7th Floor 2-21 Ageba-Cho, Shinjuku-Ku Tokyo, 162-0824, Japan Telephone: +81 3 5225 3638 Fax: +81 3 5261 8733 Active Motif China 787 Kangqiao Road Building 10, Suite 202, Pudong District Shanghai, 201315, China Telephone: (86)-21-20926090 Hotline: 400-018-8123 Copyright 2021 Active Motif, Inc. www.activemotif.com Information in this manual is subject to change without notice and does not constitute a commit- ment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any kind, either explicit or implied. Information may be changed or updated in this manual at any time. This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, in whole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and interna- tional treaties. The manufacturer of this documentation is Active Motif, Inc. © 2021 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008.
    [Show full text]
  • H-Ras Gtpase
    H-Ras GTPase Key to Understanding Cancer? Marquette University High School SMART Team: Mohammed Ayesh, Wesley Borden, Andrew Bray, Brian Digiacinto, Patrick Jordan, David Moldenhauer, Thomas Niswonger, Joseph Radke, Amit Singh, Alex Vincent, and Caleb Vogt Teachers: Keith Klestinski and David Vogt Mentor: Evgenii Kovrigin, Ph.D., Medical College of Wisconsin Abstract Cell Cycle Control The protein known as H-Ras GTPase is essential to H-ras is activated late in the G1 phase. proper biological functioning in the entire web of life. The Once H-ras is activated, the cell advances main function of this protein is giving the "stop" signal to past the G1 checkpoint and is compelled to the process of cell reproduction. Unfortunately, this protein complete mitosis. is not perfect and severe consequences, such as cancer, can arise when H-Ras GTPase malfunctions. H-Ras GTPase is a protein from the large family of enzymes that bind and split GTP. H-Ras GTPase is vital in processes like cell-to-cell communication, protein translation in ribosomes, and programmed cell death Ras GTPase Ras GDPase (apoptosis). Its main fields of operation are determining Active Inactive stem cell into specific functioning cells, as well as replicating preexisting "specialized" cells. All G domain based proteins have a universal structure and two Controlling the “Switch” between universal switch mechanisms, which consist of a mixed, six-stranded beta sheet and five alpha helices. H-Ras Active and Inactive States GTPase works by first dissociating from GDP and binding In the graphics (above and below), H-ras is shown in both © 2008, Physiomics, Corp.
    [Show full text]
  • Appendix 2. Significantly Differentially Regulated Genes in Term Compared with Second Trimester Amniotic Fluid Supernatant
    Appendix 2. Significantly Differentially Regulated Genes in Term Compared With Second Trimester Amniotic Fluid Supernatant Fold Change in term vs second trimester Amniotic Affymetrix Duplicate Fluid Probe ID probes Symbol Entrez Gene Name 1019.9 217059_at D MUC7 mucin 7, secreted 424.5 211735_x_at D SFTPC surfactant protein C 416.2 206835_at STATH statherin 363.4 214387_x_at D SFTPC surfactant protein C 295.5 205982_x_at D SFTPC surfactant protein C 288.7 1553454_at RPTN repetin solute carrier family 34 (sodium 251.3 204124_at SLC34A2 phosphate), member 2 238.9 206786_at HTN3 histatin 3 161.5 220191_at GKN1 gastrokine 1 152.7 223678_s_at D SFTPA2 surfactant protein A2 130.9 207430_s_at D MSMB microseminoprotein, beta- 99.0 214199_at SFTPD surfactant protein D major histocompatibility complex, class II, 96.5 210982_s_at D HLA-DRA DR alpha 96.5 221133_s_at D CLDN18 claudin 18 94.4 238222_at GKN2 gastrokine 2 93.7 1557961_s_at D LOC100127983 uncharacterized LOC100127983 93.1 229584_at LRRK2 leucine-rich repeat kinase 2 HOXD cluster antisense RNA 1 (non- 88.6 242042_s_at D HOXD-AS1 protein coding) 86.0 205569_at LAMP3 lysosomal-associated membrane protein 3 85.4 232698_at BPIFB2 BPI fold containing family B, member 2 84.4 205979_at SCGB2A1 secretoglobin, family 2A, member 1 84.3 230469_at RTKN2 rhotekin 2 82.2 204130_at HSD11B2 hydroxysteroid (11-beta) dehydrogenase 2 81.9 222242_s_at KLK5 kallikrein-related peptidase 5 77.0 237281_at AKAP14 A kinase (PRKA) anchor protein 14 76.7 1553602_at MUCL1 mucin-like 1 76.3 216359_at D MUC7 mucin 7,
    [Show full text]
  • Small Gtpase Ran and Ran-Binding Proteins
    BioMol Concepts, Vol. 3 (2012), pp. 307–318 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/bmc-2011-0068 Review Small GTPase Ran and Ran-binding proteins Masahiro Nagai 1 and Yoshihiro Yoneda 1 – 3, * highly abundant and strongly conserved GTPase encoding ∼ 1 Biomolecular Dynamics Laboratory , Department a 25 kDa protein primarily located in the nucleus (2) . On of Frontier Biosciences, Graduate School of Frontier the one hand, as revealed by a substantial body of work, Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Ran has been found to have widespread functions since Osaka 565-0871 , Japan its initial discovery. Like other small GTPases, Ran func- 2 Department of Biochemistry , Graduate School of Medicine, tions as a molecular switch by binding to either GTP or Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 , GDP. However, Ran possesses only weak GTPase activ- Japan ity, and several well-known ‘ Ran-binding proteins ’ aid in 3 Japan Science and Technology Agency , Core Research for the regulation of the GTPase cycle. Among such partner Evolutional Science and Technology, Osaka University, 1-3 molecules, RCC1 was originally identifi ed as a regulator of Yamada-oka, Suita, Osaka 565-0871 , Japan mitosis in tsBN2, a temperature-sensitive hamster cell line (3) ; RCC1 mediates the conversion of RanGDP to RanGTP * Corresponding author in the nucleus and is mainly associated with chromatin (4) e-mail: [email protected] through its interactions with histones H2A and H2B (5) . On the other hand, the GTP hydrolysis of Ran is stimulated by the Ran GTPase-activating protein (RanGAP) (6) , in con- Abstract junction with Ran-binding protein 1 (RanBP1) and/or the large nucleoporin Ran-binding protein 2 (RanBP2, also Like many other small GTPases, Ran functions in eukaryotic known as Nup358).
    [Show full text]
  • GTP-Binding Proteins • Heterotrimeric G Proteins
    GTP-binding proteins • Heterotrimeric G proteins • Small GTPases • Large GTP-binding proteins (e.g. dynamin, guanylate binding proteins, SRP- receptor) GTP-binding proteins Heterotrimeric G proteins Subfamily Members Prototypical effect Gs Gs, Golf cAMP Gi/o Gi, Go, Gz cAMP , K+-current Gq Gq, G11, G14, Inositol trisphosphate, G15/16 diacylglycerol G12/13 G12, G13 Cytoskeleton Transducin Gt, Gustducin cGMP - phosphodiesterase Offermanns 2001, Oncogene GTP-binding proteins Small GTPases Family Members Prototypical effect Ras Ras, Rap, Ral Cell proliferation; Cell adhesion Rho Rho, Rac, CDC42 Cell shape change & motility Arf/Sar Arf, Sar, Arl Vesicles: fission and fusion Rab Rab (1-33) Membrane trafficking between organelles Ran Ran Nuclear membrane plasticity Nuclear import/export The RAB activation-inactivation cycle REP Rab escort protein GGT geranylgeranyl-transferase GDI GDP-dissociation inhibitor GDF GDI displacementfactor Lipid e.g. RAS e.g. ARF1 modification RAB, Gi/o of GTP-binding proteins Lipid modification Enzyme Reaction Myristoylation N-myristoyl-transferase myristoylates N-terminal glycin Farnesylation Farnesyl-transferase, Transfers prenyl from prenyl-PPi Geranylgeranylation geranylgeranyltransferase to C-terminal CAAX motif Palmitoylation DHHC protein Cysteine-S-acylation General scheme of coated vesicle formation T. J. Pucadyil et al., Science 325, 1217-1220 (2009) Published by AAAS General model for scission of coated buds T. J. Pucadyil et al., Science 325, 1217-1220 (2009) Published by AAAS Conformational change in Arf1 and Sar1 GTPases, regulators of coated vesicular transport This happens if protein is trapped in the active conformation Membrane tubules formed by GTP- bound Arf1 Membrane tubules formed by GTP-bound Sar1 Published by AAAS T.
    [Show full text]
  • Small Gtpases of the Ras and Rho Families Switch On/Off Signaling
    International Journal of Molecular Sciences Review Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases Alazne Arrazola Sastre 1,2, Miriam Luque Montoro 1, Patricia Gálvez-Martín 3,4 , Hadriano M Lacerda 5, Alejandro Lucia 6,7, Francisco Llavero 1,6,* and José Luis Zugaza 1,2,8,* 1 Achucarro Basque Center for Neuroscience, Science Park of the Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), 48940 Leioa, Spain; [email protected] (A.A.S.); [email protected] (M.L.M.) 2 Department of Genetics, Physical Anthropology, and Animal Physiology, Faculty of Science and Technology, UPV/EHU, 48940 Leioa, Spain 3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, 180041 Granada, Spain; [email protected] 4 R&D Human Health, Bioibérica S.A.U., 08950 Barcelona, Spain 5 Three R Labs, Science Park of the UPV/EHU, 48940 Leioa, Spain; [email protected] 6 Faculty of Sport Science, European University of Madrid, 28670 Madrid, Spain; [email protected] 7 Research Institute of the Hospital 12 de Octubre (i+12), 28041 Madrid, Spain 8 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain * Correspondence: [email protected] (F.L.); [email protected] (J.L.Z.) Received: 25 July 2020; Accepted: 29 August 2020; Published: 31 August 2020 Abstract: Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs).
    [Show full text]
  • RBD11, a Bioengineered Rab11-Binding Module for Visualizing and Analyzing Endogenous Rab11
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428185; this version posted January 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11 Futaba Osaki1, Takahide Matsui1,*, Shu Hiragi1, Yuta Homma1, and Mitsunori Fukuda1,2,* 1Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan Running title: Development of RBD11, a specific Rab11-binding module 2Lead Contact *Correspondence: Dr. Takahide Matsui, [email protected]; Tel.: +81-22-795-3641; Fax: +81-22-795-3642 Dr. Mitsunori Fukuda, [email protected]; Tel.: +81-22-795-7731; Fax: +81-22-795-7733 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428185; this version posted January 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT The small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25.
    [Show full text]
  • Subassembly of the Herpes Simplex Virus 1 Helicase-Primase Composed of the UL5 and UL52 Gene Products
    Proc. Nati. Acad. Sci. USA Vol. 88, pp. 1105-1109, February 1991 Biochemistry Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products MARK S. DODSON AND I. R. LEHMAN Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305 Contributed by I. R. Lehman, November 5, 1990 ABSTRACT Herpes simplex virus 1 encodes a helicase- Enzymatic Assays and Gel Electrophoresis. DNA-depen- primase that is composed of the products ofthe UL5, UL8, and dent nucleoside triphosphatase assays were performed as UL52 genes. A stable subassembly consisting of only the UL5 described (2, 8). One unit of enzyme hydrolyzes 1.0 nmol of and UL52 gene products has been purified to near homogeneity nucleoside triphosphate per hr (2). The coupled primase from insect cells doubly infected with baculovirus recombinant assay was used to follow primase activity during purification for these two genes. The purified subassembly has the DNA- of the UL5/UL52 subassembly (3, 9). One unit of primase dependent ATPase, DNA-dependent GTPase, DNA helicase, incorporates 1.0 pmol of [3H]dATP per hr (3). DNA helicase and DNA primase activities that are characteristic of the assays were performed as described (2). The unwound prod- three-subunit holoenzyme. The purified UL8 gene product, ucts were visualized by autoradiography and quantitated by although required for viral DNA replication, neither exhibits scanning with a Molecular Dynamics 300A computing den- these enzymatic activities nor stably associates with either the sitometer. SDS/polyacrylamide gel electrophoresis and sil- UL5 or the UL52 gene product.
    [Show full text]
  • Structural Analysis of the Ras Gtpase Activating Protein Catalytic Domain by Semirandom Mutagenesis: Implications for a Mechanism of Interaction with Ras-GTP1
    [CANCERRESEARCH54. 5438-5444, October 15, 1994] Structural Analysis of the Ras GTPase Activating Protein Catalytic Domain by Semirandom Mutagenesis: Implications for a Mechanism of Interaction with Ras-GTP1 Lisa Hettich and Mark Marshall2 Department of Medicine, Division of Hematology/Oncology. Indiana UniversüySchool of Medicine and Waither Oncology Center, Indianapolis. indiana 46202 ABSTRACT region of GAP in the regulation of cytoskeletal structure and cell adhe sion to the extracellular matrix (14). Expression of the GAP NH2- The bovine complementary DNA encoding the catalytic domain of Ras terminal SH2-SH3-SH2 region in Rat-2 fibroblasts resulted in a disrup GTPase activating protein was mutagenized semirandomly using a van tionofactinstressfibersandfocalcontacts.Thiseffectisprobablycaused atlon of the polymerase chain reaction. Sixty-four mutated codons were identified with seventeen of the mutations deleterious to Ras GTPase by the action of the GAP-associated protein, p190, and Rho (15-17). activating function. All of the inactivating single mutations affected the Changes in the cytoskeleton and loss of adhesion are important compo structure of the catalytic fragment as assessed by large decreases in nents of cell growth. soluble protein when expressed in Escherichia coli. Upon exnmlnation of In addition to its potential as a Ras effector molecule, GAP is the Ran binding properties of 10 of the mutants, only 1 was measurably currently the best model for examining GTP-dependent protein inter Impaired for I(as binding and 4 appeared to have increased affinity for actions with Ras. Ras-GTP has been shown to bind to GAP, while Ran. These results demonstrate that Ras binding and GTPase activation Ras-GDP does not (18).
    [Show full text]
  • Small G-Protein They Bind GTP, and “OFF” After the GTP Has Been Hydrolyzed to GDP (Which in Turn Remains Bound)
    MONOMERIC G-PROTEINS (GTPases) In addition to heterotrimeric G-proteins, there is a superfamily of small monomeric G-proteins. Their size is around 21 kDa, approximately one half of the average size of the subunits of heterotrimeric proteins. These small proteins also operate as switches, which are “ON” when Small G-protein they bind GTP, and “OFF” after the GTP has been hydrolyzed to GDP (which in turn remains bound). There is a larger family of small GTP-binding switch proteins, related to G Monomeric G-proteins are activated by proteins which induce a conformational change resulting in reduced affinity to GDP, and thus in G-protein : heterotrimeric G proteins GDP release. The general term for such proteins is GEF (guanine nucleotide exchange factor). The GEFs of the small G-proteins are not the activated receptors, but rather proteins downstream the signaling cascade. GEFs vary with regard to their mode of activation and their selectivity for specific monomeric G-proteins. Small GTP-binding proteins include (roles indicated): initiation & elongation factors (protein synthesis). Compared to the subunit of heterotrimeric G-proteins, GTPase activity Ras (growth factor signal cascades). of the monomeric G-proteins is very low in the absence of interference. Rab (vesicle targeting and fusion). However, association with a protein of the GAP (GTPase-activating ARF (forming vesicle coatomer coats). protein) type results in very rapid GTP hydrolysis. Ran (transport of proteins into & out of the nucleus). As in the case of GEFs, the activation state of GAPs can be regulated, and Rho (regulation of actin cytoskeleton) GAPs are selective with regard to the small G-proteins they affect.
    [Show full text]
  • Directing Gene Expression to Gustducin-Positive Taste Receptor Cells
    The Journal of Neuroscience, July 15, 1999, 19(14):5802–5809 Directing Gene Expression to Gustducin-Positive Taste Receptor Cells Gwendolyn T. Wong, Luis Ruiz-Avila, and Robert F. Margolskee Howard Hughes Medical Institute, Department of Physiology and Biophysics, The Mount Sinai School of Medicine, New York, New York 10029 We have demonstrated that an 8.4 kb segment (GUS8.4 )from Expression of the lacZ transgene from the GUS8.4 promoter and the upstream region of the mouse a-gustducin gene acts as a of endogenous gustducin was coordinately lost after nerve fully functional promoter to target lacZ transgene expression to section and simultaneously recovered after reinnervation, con- the gustducin-positive subset of taste receptor cells (TRCs). firming the functionality of this promoter. Transgenic expression a The GUS8.4 promoter drove TRC expression of the of rat -gustducin restored responsiveness of gustducin null b-galactosidase marker at high levels and in a developmentally mice to both bitter and sweet compounds, demonstrating the appropriate pattern. The gustducin minimal 1.4 kb promoter utility of the gustducin promoter. (GUS1.4 ) by itself was insufficient to specify TRC expression. We also identified an upstream enhancer from the distal portion Key words: gustducin; taste receptor cells; guanine nucleo- of the murine gustducin gene that, in combination with the tide binding regulatory proteins; gustation; transgenic mice; minimal promoter, specified TRC expression of transgenes. promoter identification; b-galactosidase Current models of taste perception in humans suggest four or five sensory cells of the stomach, duodenum, and pancreas (Ho¨fer et primary taste submodalities: sweet, bitter, salty, sour, and umami al., 1996; Ho¨fer and Drenckhahn, 1998).
    [Show full text]
  • RAB11-Mediated Trafficking and Human Cancers: an Updated Review
    biology Review RAB11-Mediated Trafficking and Human Cancers: An Updated Review Elsi Ferro 1,2, Carla Bosia 1,2 and Carlo C. Campa 1,2,* 1 Department of Applied Science and Technology, Politecnico di Torino, 24 Corso Duca degli Abruzzi, 10129 Turin, Italy; [email protected] (E.F.); [email protected] (C.B.) 2 Italian Institute for Genomic Medicine, c/o IRCCS, Str. Prov. le 142, km 3.95, 10060 Candiolo, Italy * Correspondence: [email protected] Simple Summary: The small GTPase RAB11 is a master regulator of both vesicular trafficking and membrane dynamic defining the surface proteome of cellular membranes. As a consequence, the alteration of RAB11 activity induces changes in both the sensory and the transduction apparatuses of cancer cells leading to tumor progression and invasion. Here, we show that this strictly depends on RAB110s ability to control the sorting of signaling receptors from endosomes. Therefore, RAB11 is a potential therapeutic target over which to develop future therapies aimed at dampening the acquisition of aggressive traits by cancer cells. Abstract: Many disorders block and subvert basic cellular processes in order to boost their pro- gression. One protein family that is prone to be altered in human cancers is the small GTPase RAB11 family, the master regulator of vesicular trafficking. RAB11 isoforms function as membrane organizers connecting the transport of cargoes towards the plasma membrane with the assembly of autophagic precursors and the generation of cellular protrusions. These processes dramatically impact normal cell physiology and their alteration significantly affects the survival, progression and metastatization as well as the accumulation of toxic materials of cancer cells.
    [Show full text]