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Pertussis toxin induces structural changes in G alpha proteins independently of ADP-ribosylation
1Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Abstract:
Pertussis toxin catalyzes ADP-ribosylation of a family of GTP-binding proteins (G alpha proteins) involved in signal transduction. It is thought that this activity is responsible for the attenuating effects of the toxin on the actions of a number of hormones and neurotransmitters. By utilizing specific antisera for detecting on electrophoretic transfer blots (Western blots) alpha proteins that are subject to ADP-ribosylation, it was found that treatment of these proteins with pertussis toxin resulted in shifts in their electrophoretic mobility and marked enhancement of their immunoreactivity compared to untreated proteins. No changes in mobility or immunoreactivity with specific antisera were observed with beta subunits of G proteins. Both effects on alpha proteins required the same ingredients, including detergents, ATP, and sulfhydryl reducing agents, that other studies have shown are required for activation of the ADP-ribosylating activity of pertussis toxin. However, NAD+, the substrate for ADP-ribosylating activity, was not required. Moreover, inhibition of the ADP-ribosylating activity by 50 mM nicotinamide failed to block the NAD-independent effects of the toxin. These findings indicate that the toxin induces structural changes in alpha proteins independently of its ADP-ribosylating activity and raise the possibility that these structural changes are primary to ADP-ribosylation and causative of many of the biological effects of pertussis toxin.
Insights
Pertussis toxin alters GTP-binding proteins (G alpha proteins) structurally, independent of its ADP-ribosylation activity. These toxin-induced changes may explain its biological effects on hormone and neurotransmitter signaling.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Pertussis toxin affects signal transduction by modifying GTP-binding proteins (G alpha proteins).
- This modification is believed to mediate the toxin's attenuating effects on hormones and neurotransmitters.
Purpose of the Study:
- To investigate the structural changes induced by pertussis toxin in G alpha proteins.
- To determine if these structural changes are dependent on the toxin's ADP-ribosylation activity.
Main Methods:
- Western blotting with specific antisera to detect ADP-ribosylated alpha and beta subunits of G proteins.
- Analysis of electrophoretic mobility and immunoreactivity shifts after toxin treatment.
- Assessing the requirement of NAD+ and nicotinamide for toxin-induced effects.
Main Results:
- Pertussis toxin treatment caused shifts in electrophoretic mobility and enhanced immunoreactivity of alpha proteins.
- No changes were observed in beta subunits.
- These effects occurred independently of NAD+ and were not blocked by nicotinamide, indicating an NAD-independent mechanism.
Conclusions:
- Pertussis toxin induces structural alterations in G alpha proteins independent of its ADP-ribosylation activity.
- These NAD-independent structural changes may be the primary event leading to ADP-ribosylation and the toxin's biological effects.