Pasteurella multocida Toxin Activates the Inositol Triphosphate Signaling Pathway in Xenopus Oocytes via Gqα-coupled Phospholipase C-β1

doi:10.1074/jbc.272.2.1268

Pasteurella multocida Toxin Activates the Inositol Triphosphate Signaling Pathway in Xenopus Oocytes via G_qα-coupled Phospholipase C-β1*

From the ^‡ Departments of Biochemistry and Molecular Biology and
^¶ Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435

^§ To whom correspondence should be addressed. Tel.: 937-775-4803; Fax: 937-775-3730; E-mail: bwilson{at}desire.wright.edu

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/11α. Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of G_qα in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G_qα cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G_qα.

Footnotes

↵* This work was supported in part by an Ohio Research Challenge Grant and Grant AI38396 from the National Institutes of Health/NIAID (to B. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PMT

Pasteurella multocida toxin

PLC

phospholipase C

PIP₂

phosphatidylinositol 4,5-bisphosphate

IP₃

inositol 1,4,5-triphosphate

PT

pertussis toxin

G-protein

GTP-binding protein

GDPβS

guanosine 5′-O-(2-(thio)diphosphate).
↵²B. A. Wilson and X. Zhu, unpublished observations.
- Received October 17, 1996.