Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Gαi and Gαo Deactivation

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits speeding deactivation. Gα deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Gα accompanying GTP hydrolysis and Gα deactivation on the millisecond time scale. Basal k cat values for Gαo, Gαi1, and Gαi2 at 20 °C were similar (0.025–0.033 s−1). GlutathioneS-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305–453) enhanced the rate of Gα deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s−1 at 3 μm, giving a catalytic efficiency of 1.7–2.8 × 106 m −1 s−1 for all three Gα subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 × 106 m −1s−1 toward Gαo, Gαi2, and Gαi1, respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Gα subunits tested, but it is specific for Gαo over Gαi1 or Gαi2. Furthermore, the specificity of RGS7 for Gαo does not depend on N- or C-terminal extensions or a Gβ5 subunit but resides in the RGS domain itself.

[1]  R. Neubig,et al.  A Point Mutation in Gαo and Gαi1Blocks Interaction with Regulator of G Protein Signaling Proteins* , 1998, The Journal of Biological Chemistry.

[2]  S. Sprang,et al.  Structure of RGS4 Bound to AlF4 −-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis , 1997, Cell.

[3]  K. Blumer,et al.  Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family , 1996, Nature.

[4]  T. Wieland,et al.  Regulators of G-protein signalling: a novel protein family involved in timely deactivation and desensitization of signalling via heterotrimeric G proteins , 1999, Naunyn-Schmiedeberg's Archives of Pharmacology.

[5]  R. Cerione,et al.  Rhodopsin-stimulated activation-deactivation cycle of transducin: kinetics of the intrinsic fluorescence response of the alpha subunit. , 1990, Biochemistry.

[6]  M. Ichikawa,et al.  RGS7 and RGS8 Differentially Accelerate G Protein-mediated Modulation of K+ Currents* , 1999, The Journal of Biological Chemistry.

[7]  R. Neubig,et al.  Fluorescent guanine nucleotide analogs and G protein activation. , 1994, The Journal of biological chemistry.

[8]  H. Dohlman,et al.  Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[9]  A. Gilman,et al.  G proteins: transducers of receptor-generated signals. , 1987, Annual review of biochemistry.

[10]  M. Farquhar,et al.  The regulator of G protein signaling family. , 2000, Annual review of pharmacology and toxicology.

[11]  V. Slepak,et al.  Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins , 1999 .

[12]  J. Thorner,et al.  RGS Proteins and Signaling by Heterotrimeric G Proteins* , 1997, The Journal of Biological Chemistry.

[13]  A. Gilman,et al.  The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. , 1982, The Journal of biological chemistry.

[14]  Seong-Woo Jeong,et al.  Endogenous Regulator of G-Protein Signaling Proteins Modify N-Type Calcium Channel Modulation in Rat Sympathetic Neurons , 2000, The Journal of Neuroscience.

[15]  R. Neubig,et al.  Roles of G(o)alpha tryptophans in GTP hydrolysis, GDP release, and fluorescence signals. , 1998, Biochemistry.

[16]  V. Slepak,et al.  Identification of the Gβ5-RGS7 Protein Complex in the Retina☆ , 1998 .

[17]  P. Chardin,et al.  Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding. , 1993, The EMBO journal.

[18]  R. Cerione,et al.  The intrinsic fluorescence of the alpha subunit of transducin. Measurement of receptor-dependent guanine nucleotide exchange. , 1988, The Journal of biological chemistry.

[19]  D. Clapham,et al.  G protein beta gamma subunits. , 1997, Annual review of pharmacology and toxicology.

[20]  N. Artemyev,et al.  Substitution of Transducin Ser202 by Asp Abolishes G-protein/RGS Interaction* , 1998, The Journal of Biological Chemistry.

[21]  S. Mumby,et al.  Myristoylation of G-protein alpha subunits. , 1994 .

[22]  A. Gilman,et al.  The effect of activating ligands on the intrinsic fluorescence of guanine nucleotide-binding regulatory proteins. , 1987, The Journal of biological chemistry.

[23]  A. Gilman,et al.  RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  H. Horvitz,et al.  EGL-10 Regulates G Protein Signaling in the C. elegans Nervous System and Shares a Conserved Domain with Many Mammalian Proteins , 1996, Cell.

[25]  N. Artemyev,et al.  Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins* , 1998, The Journal of Biological Chemistry.

[26]  H. Lester,et al.  RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels , 1997 .

[27]  A. Gilman,et al.  GAIP and RGS4 Are GTPase-Activating Proteins for the Gi Subfamily of G Protein α Subunits , 1996, Cell.

[28]  E M Ross,et al.  Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  L. Jan,et al.  G-protein signaling: Fine-tuning signaling kinetics , 1998, Current Biology.

[30]  A. Gilman,et al.  Regulators of G Protein Signaling 6 and 7 , 1999, The Journal of Biological Chemistry.

[31]  A. Gilman,et al.  Mammalian RGS Proteins: Barbarians at the Gate* , 1998, The Journal of Biological Chemistry.

[32]  M. Koelle.  A new family of G-protein regulators - the RGS proteins. , 1997, Current opinion in cell biology.

[33]  J. Hepler.  Emerging roles for RGS proteins in cell signalling. , 1999, Trends in pharmacological sciences.

[34]  T. Kozasa,et al.  The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. K. Chan,et al.  Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones , 1982, Molecular and cellular biology.

[36]  K. Blumer,et al.  RGS family members: GTPase-activating proteins for heterotrimeric G-protein α-subunits , 1996, Nature.

[37]  J. Thorner,et al.  Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit) , 1996, Molecular and cellular biology.

[38]  O. Andreassen,et al.  Mice Deficient in Cellular Glutathione Peroxidase Show Increased Vulnerability to Malonate, 3-Nitropropionic Acid, and 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine , 2000, The Journal of Neuroscience.

[39]  A. Gilman,et al.  The effect of GTP and Mg2+ on the GTPase activity and the fluorescent properties of Go. , 1987, The Journal of biological chemistry.

[40]  N. Artemyev,et al.  Interaction of human retinal RGS with G‐protein α‐subunits , 1997 .

[41]  S R Sprang,et al.  G protein mechanisms: insights from structural analysis. , 1997, Annual review of biochemistry.

[42]  M. Farquhar,et al.  RGS proteins: more than just GAPs for heterotrimeric G proteins. , 1999 .

[43]  M. Cockett,et al.  RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells , 1998, Journal of neurochemistry.

[44]  A. Gilman,et al.  The GTPase-activating Protein RGS4 Stabilizes the Transition State for Nucleotide Hydrolysis* , 1996, The Journal of Biological Chemistry.

[45]  T. Hunt,et al.  RGS10 is a selective activator of Gαi GTPase activity , 1996, Nature.