Related Experiment Videos
Pertussis toxin mediates ADP-ribosylation of pituitary membrane proteins
Abstract:
Pertussis toxin catalyzes the ADP-ribosylation of the inhibitory subunit (Ni) of adenylate cyclase. Despite several studies which demonstrate that pertussis toxin influences cyclic AMP accumulation and hormone secretion in normal anterior pituitary cells, the target protein(s) for this toxin in these cells has not been identified. We have examined pertussis toxin mediated ADP-ribosylation in membrane preparations of tumor-derived (235-1, GH4C1, GH3) and normal anterior pituitary cells. Autoradiograms of SDS gels reveal that in the presence of [32P]NAD and pertussis toxin, a 40 kilodalton protein band was labeled in membrane preparations from cells cultured with vehicle. Such labeling was diminished when the cells were exposed to pertussis toxin (35 ng/ml) for 18 hours. Similar results were found in both tumor-derived and normal (monkey and rat) anterior pituitary cells. The pertussis toxin specific band was further resolved into two bands of approximately 39 and 41 kilodaltons. Autoradiograms of two dimensional gels revealed two ADP-ribosylated spots with isoelectric points of 5.7 and 6.2, although the molecular weights appeared identical (approx. 40 kilodaltons). Cholera toxin, which catalyzes the ADP-ribosylation of a 45 kilodalton protein did not prevent labeling of the pertussis toxin-specific band(s) in cells pretreated with cholera toxin. These results suggest that pertussis toxin specifically mediates ADP-ribosylation of the Ni protein in normal anterior pituitary cells.
Insights
Pertussis toxin specifically targets and modifies the inhibitory subunit (Ni) of adenylate cyclase in anterior pituitary cells. This study identifies the Ni protein as the primary ADP-ribosylation target for pertussis toxin in these cells.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Signaling
Background:
- Pertussis toxin (PT) is known to affect cyclic AMP levels and hormone secretion in pituitary cells.
- The specific protein targets of PT in anterior pituitary cells remain unidentified.
- PT catalyzes ADP-ribosylation, a key post-translational modification.
Purpose of the Study:
- To identify the specific protein targets of pertussis toxin in anterior pituitary cells.
- To investigate the ADP-ribosylation patterns mediated by PT in both normal and tumor-derived pituitary cells.
- To confirm the role of the inhibitory subunit of adenylate cyclase (Ni) as a PT target.
Main Methods:
- Utilized SDS-PAGE and two-dimensional gel electrophoresis to analyze protein labeling.
- Employed [32P]NAD and pertussis toxin to detect ADP-ribosylation in cell membrane preparations.
- Compared PT-mediated ADP-ribosylation with cholera toxin-mediated ADP-ribosylation.
Main Results:
- A 40 kDa protein band was specifically labeled by PT in anterior pituitary cell membranes.
- This labeling was reduced upon prolonged exposure to PT.
- Two distinct ADP-ribosylated spots (pI 5.7 and 6.2) were observed, corresponding to the ~40 kDa protein.
- Cholera toxin did not affect PT-specific labeling, indicating distinct targets.
Conclusions:
- Pertussis toxin specifically mediates ADP-ribosylation of the Ni protein in anterior pituitary cells.
- The identified 40 kDa protein is likely the Ni subunit of adenylate cyclase.
- These findings clarify the molecular mechanism of PT action in pituitary endocrine regulation.