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Point-mutated p21ras couples a muscarinic receptor to calcium channels and polyphosphoinositide hydrolysis
Abstract:
A high-affinity muscarinic receptor is detectable both in normal 3T3 mouse fibroblasts and in their transformed counterpart obtained by transfection with the oncogene EJ/T24-H-ras. However, only the transformed cell line is responsive to muscarinic agonist carbamylcholine in terms of Ca2+ influx and polyphosphoinositide hydrolysis, whereas the normal cell line is unresponsive. Using a point-mutated p21ras protein and monoclonal antibodies anti-p21ras, we provide evidences that p21ras couples to receptor-operating calcium channels and to polyphosphoinositide hydrolysis a muscarinic receptor which is uncoupled in normal mouse fibroblasts.
Insights
Transformed mouse fibroblasts exhibit muscarinic receptor signaling, unlike normal cells. The oncogene p21ras protein enables this response, coupling the receptor to calcium influx and cell signaling pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- Muscarinic receptors are G protein-coupled receptors involved in various cellular processes.
- Ras proteins are key regulators of cell signaling pathways, often implicated in cancer development.
Purpose of the Study:
- To investigate the role of the oncogene p21ras in muscarinic receptor signaling in fibroblasts.
- To determine why normal fibroblasts do not respond to muscarinic agonists while transformed cells do.
Main Methods:
- Utilized normal and transformed 3T3 mouse fibroblasts.
- Employed muscarinic agonist carbamylcholine to stimulate cells.
- Assessed calcium (Ca2+) influx and polyphosphoinositide hydrolysis.
- Used point-mutated p21ras protein and anti-p21ras monoclonal antibodies for mechanistic studies.
Main Results:
- Both normal and transformed fibroblasts express high-affinity muscarinic receptors.
- Transformed cells, but not normal cells, showed a response to carbamylcholine, including Ca2+ influx and polyphosphoinositide hydrolysis.
- Evidence suggests p21ras couples muscarinic receptors to calcium channels and polyphosphoinositide hydrolysis in transformed cells.
Conclusions:
- The oncogene p21ras is essential for coupling muscarinic receptors to downstream signaling pathways in fibroblasts.
- Aberrant p21ras activity in transformed cells activates previously uncoupled muscarinic receptors.
- This study elucidates a novel mechanism linking ras oncogenes to G protein-coupled receptor signaling.