Activation of Gαi and Subsequent Uncoupling of Receptor-Gαi Signaling by Pasteurella multocida Toxin*

Bacterial protein toxins are powerful tools for elucidating signaling mechanisms in eukaryotic cells. A number of bacterial protein toxins, e.g. cholera toxin, pertussis toxin (PTx), or Pasteurella multocida toxin (PMT), target heterotrimeric G proteins and have been used to stimulate or block specific signaling pathways or to demonstrate the contribution of their target proteins in cellular effects. PMT is a major virulence factor of P. multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PMT modulates various signaling pathways, including phospholipase Cβ and RhoA, by acting on the heterotrimeric G proteins Gαq and Gα12/13, respectively. Here we report that PMT is a powerful activator of Gi protein. We show that PMT decreases basal isoproterenol and forskolin-stimulated cAMP accumulation in intact Swiss 3T3 cells, inhibits adenylyl cyclase activity in cell membrane preparations, and enhances the inhibition of cAMP accumulation caused by lysophosphatidic acid via endothelial differentiation gene receptors. PMT-mediated inhibition of cAMP production is independent of toxin activation of Gαq and/or Gα12/13. Although the effects of PMT are not inhibited by PTx, PMT blocks PTx-catalyzed ADP-ribosylation of Gi. PMT also inhibits steady-state GTPase activity and GTP binding of Gi in Swiss 3T3 cell membranes stimulated by lysophosphatidic acid. The data indicate that PMT is a novel activator of Gi, modulating its GTPase activity and converting it into a PTx-insensitive state.

[1]  B. Nürnberg.  Pertussis Toxin as a Cell Biology Tool , 2008 .

[2]  B. Matthews.  Changes at Protein Science , 2008 .

[3]  G. Milligan,et al.  Action of Pasteurella multocida toxin on Galpha(q) is persistent and independent of interaction with G-protein-coupled receptors. , 2007, Cellular signalling.

[4]  Masami Miyake,et al.  Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin , 2007, Proceedings of the National Academy of Sciences.

[5]  K. Aktories,et al.  Modulation of Host Cell Gene Expression through Activation of STAT Transcription Factors by Pasteurella multocida Toxin* , 2007, Journal of Biological Chemistry.

[6]  B. Wilson,et al.  CHAPTER 22 – Pasteurella multocida toxin , 2006 .

[7]  K. Aktories,et al.  Pasteurella multocida Toxin-induced Activation of RhoA Is Mediated via Two Families of Gα Proteins, Gαq and Gα12/13* , 2005, Journal of Biological Chemistry.

[8]  M. Taniguchi,et al.  A Novel Gαq/11-selective Inhibitor* , 2004, Journal of Biological Chemistry.

[9]  K. Aktories,et al.  Action of Pasteurella multocida Toxin Depends on the Helical Domain of Gαq* , 2004, Journal of Biological Chemistry.

[10]  B. Wilson,et al.  Pasteurella multocida toxin as a tool for studying Gq signal transduction. , 2004, Reviews of physiology, biochemistry and pharmacology.

[11]  G. Schultz,et al.  Receptor-dependent RhoA Activation in G12/G13-deficient Cells , 2003, Journal of Biological Chemistry.

[12]  K. Aktories,et al.  His1205 and His1223 are essential for the activity of the mitogenic Pasteurella multocida toxin. , 2003, Biochemistry.

[13]  G. Milligan.  Principles: extending the utility of [35S]GTP gamma S binding assays. , 2003, Trends in pharmacological sciences.

[14]  R. Sowdhamini,et al.  Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida , 2001, Infection and Immunity.

[15]  K. Aktories,et al.  Biological Activity of a C-Terminal Fragment ofPasteurella multocida Toxin , 2001, Infection and Immunity.

[16]  G. Schultz,et al.  Pleiotropic Effects of Pasteurella multocida Toxin Are Mediated by Gq-dependent and -independent Mechanisms , 2001, The Journal of Biological Chemistry.

[17]  A. Grigoriadis,et al.  Pasteurella multocida toxin: the mitogenic toxin that stimulates signalling cascades to regulate growth and differentiation. , 2001, International journal of medical microbiology : IJMM.

[18]  B. Wilson,et al.  Differential Modulation and Subsequent Blockade of Mitogenic Signaling and Cell Cycle Progression by Pasteurella multocida Toxin , 2000, Infection and Immunity.

[19]  D. Ladant,et al.  The comprehensive sourcebook of bacterial protein toxins , 1999 .

[20]  A. Miles,et al.  Activity of the Mitogenic Pasteurella multocida Toxin Requires an Essential C-Terminal Residue , 1998, Infection and Immunity.

[21]  Melvin I. Simon,et al.  Embryonic cardiomyocyte hypoplasia and craniofacial defects in Gαq. Gα11‐mutant mice , 1998 .

[22]  E. Akam,et al.  Pharmacological characterization of type 1α metabotropic glutamate receptor‐stimulated [35S]‐GTPγS binding , 1997 .

[23]  P. Casey,et al.  Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins. , 1997, The Biochemical journal.

[24]  B. Wilson,et al.  Pasteurella multocida Toxin Activates the Inositol Triphosphate Signaling Pathway in Xenopus Oocytes via Gqα-coupled Phospholipase C-β1* , 1997, The Journal of Biological Chemistry.

[25]  K. Aktories.  Bacterial toxins : tools in cell biology and pharmacology , 1997 .

[26]  P. Mullan,et al.  Pasteurella multocida toxin is a mitogen for bone cells in primary culture , 1996, Infection and immunity.

[27]  S. Okuya,et al.  Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer but not its free alpha-subunit. , 1994, Biochemistry.

[28]  G. Schultz,et al.  G12 and G13 alpha-subunits are immunochemically detectable in most membranes of various mammalian cells and tissues. , 1994, Biochemical and biophysical research communications.

[29]  R. A. Johnson,et al.  Determination of adenylyl cyclase catalytic activity using single and double column procedures. , 1994, Methods in enzymology.

[30]  D. Huhn,et al.  Incomplete functional differentiation of HL‐60 leukemic cells by synthetic lipopetides , 1992 .

[31]  J. Gil,et al.  Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation , 1991, The Journal of cell biology.

[32]  A. C. Murphy,et al.  Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells. , 1991, The Journal of biological chemistry.

[33]  J. Fain.  G Protein Involvement in Signal Transduction , 1991 .

[34]  J. Moss,et al.  Adp-Ribosylating Toxins and G Proteins: Insights into Signal Transduction , 1990 .

[35]  E. Rozengurt,et al.  Pasteurella multocida toxin: potent mitogen for cultured fibroblasts. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[36]  E. Ross,et al.  Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). , 1988, The Journal of biological chemistry.

[37]  P. Casey,et al.  G protein involvement in receptor-effector coupling. , 1988, The Journal of biological chemistry.

[38]  T. Katada,et al.  Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities. , 1986, The Journal of biological chemistry.

[39]  M. Ui,et al.  Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP ribosylation by islet-activating protein, pertussis toxin, in adipocyte membranes. , 1983, The Journal of biological chemistry.

[40]  T. Katada,et al.  Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. , 1982, Proceedings of the National Academy of Sciences of the United States of America.