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

• Published in: The Journal of biological chemistry Vol. 272; no. 2; p. 1268
• Main Authors: Brenda A. Wilson, Xinjun Zhu, Mengfei Ho, Luo Lu
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 10.01.1997
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.2.1268

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 Ca 2+ -dependent Cl − current. Injection of PMT induced an inward, two-component Cl − current, similar to that evoked by injection of IP 3 through intracellular Ca 2+ mobilization and Ca 2+ influx through voltage-gated Ca 2+ channels. These PMT-induced currents were blocked by specific inhibitors of Ca 2+ and Cl − channels, removal of extracellular Ca 2+ , or chelation of intracellular Ca 2+ . 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.