Pasteurella multocida toxin activates the inositol triphosphate signaling pathway in Xenopus oocytes via G(q)α-coupled phospholipase C-β1

Brenda A. Wilson, Xinjun Zhu, Mengfei Ho, Luo Lu

Research output: Contribution to journalArticlepeer-review

Abstract

Pasteurella multocida toxin (PMT) has been hypothesized to cause activation of a GTP-binding protein (G-protein)-coupled phosphatidylinositol- specific phospholipase C (PLC) in intact cells. We used voltage-clamped Xenopus oocytes to test for direct PMT-mediated stimulation of PLC by monitoring the endogenous Ca2+-dependent Cl- current. Injection of PMT induced an inward, two-component Cl- current, similar to that evoked by injection of IP3 through intracellular Ca2+ mobilization and Ca2+ influx through voltage-gated Ca2+ channels. These PMT-induced currents were blocked by specific inhibitors of Ca2+ and Cl- channels, removal of extracellular Ca2+, or chelation of intracellular Ca2+. Specific antibodies directed against an N-terminal, but not a C-terminal, peptide of PMT inhibited the toxin-induced currents, implicating that the N terminus of PMT is important for toxin activity. Injection with specific antibodies against PLCβ1, PLCβ2, PLCβ3, or PLCγ1 identified PLCβ1 as the primary mediator of the PMT-induced Cl- currents. Injection with guanosine 5'-O-(2- (thio)diphosphate), antibodies to the common GTP-binding region of G-protein α subunits, or antibodies to different regions of G-protein β subunits established the involvement of a G-protein α subunit in PMT-activation of PLCβ1. Injection with specific antibodies against the α-subunits of G(q/11), G(s/olf) G(i/o/t/z), or G(i-1/i-2/i-3) isoforms confirmed the involvement of G(q/11)α. Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of G(q)α in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G(q)α cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G(q)α.

Original languageEnglish (US)
Pages (from-to)1268-1275
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number2
DOIs
StatePublished - 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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