Pasteurella multocida toxin activates the inositol triphosphate signaling pathway in Xenopus oocytes via G(q)alpha-coupled phospholipase C-beta1.
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 C1- 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 PLCbeta1, PLCbeta2, PLCbeta3, or PLCgamma1 identified PLCbeta1 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 alpha subunits, or antibodies to different regions of G-protein beta subunits established the involvement of a G-protein alpha subunit in PMT-activation of PLCbeta1. Injection with specific antibodies against the alpha-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 Gq/11alpha. Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of G(q)alpha in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G(q)alpha cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G(q)alpha.
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Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA. bwilson@desire.wright.edu
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D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D016979:Pasteurella multocida; D037342:Pertussis Toxin; D054799:Phospholipase C beta; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014981:Xenopus
D000906:Antibodies; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018118:Chloride Channels; D007527:Isoenzymes; C062258:Pasteurella multocida toxin; D010566:Virulence Factors, Bordetella; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D002118:Calcium