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A C-terminal mutant of the G protein beta subunit deficient in the activation of phospholipase C-beta
1Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
The molecular mechanism by which the G protein betagamma complex modulates multiple mammalian effector pathways is unknown. Homolog-scanning mutagenesis of the G protein beta subunit was employed to identify residues critical for the activation of phospholipase C-beta2 (PLC-beta2). A series of chimeras was made by introducing small segments of the Dictyostelium beta subunit into a background of mammalian beta1 and tested in COS cell cotransfection assays for their ability to activate PLC-beta2 and assemble with mammalian gamma2. A chimera that contained four Dictyostelium beta substitutions within the C-terminal 14 residues was unable to activate PLC-beta2 when cotransfected with gamma, despite its demonstrable expression in a gamma-dependent manner. Cotransfection of the mutant blocked m2 muscarinic receptor activation of PLC by a pertussis toxin-sensitive pathway. This C-terminal mutant retained the ability, however, to stimulate the mitogen-activated protein kinase pathway. These results imply that activation of different betagamma-responsive effectors is mediated by distinct domains.
Insights
The G protein betagamma complex uses distinct domains to activate different cellular pathways. This study identified specific residues in the G protein beta subunit critical for phospholipase C-beta2 activation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- The G protein betagamma complex is a key regulator of multiple mammalian effector pathways.
- The precise molecular mechanisms underlying betagamma complex signaling remain largely unknown.
- Identifying specific domains involved in effector pathway modulation is crucial for understanding cell signaling.
Purpose of the Study:
- To elucidate the molecular mechanism by which the G protein betagamma complex modulates effector pathways.
- To identify critical residues within the G protein beta subunit responsible for activating phospholipase C-beta2 (PLC-beta2).
Main Methods:
- Homolog-scanning mutagenesis of the G protein beta subunit.
- Construction of chimeric G protein beta subunits using Dictyostelium and mammalian sequences.
- COS cell cotransfection assays to assess PLC-beta2 activation and complex assembly with gamma2.
- Pertussis toxin-sensitive pathway analysis and mitogen-activated protein kinase pathway stimulation assays.
Main Results:
- A G protein beta subunit chimera with substitutions in the C-terminal 14 residues failed to activate PLC-beta2.
- This C-terminal mutant demonstrated gamma-dependent expression but blocked m2 muscarinic receptor-mediated PLC activation.
- The mutant retained its ability to stimulate the mitogen-activated protein kinase pathway.
Conclusions:
- Distinct domains of the G protein beta subunit mediate the activation of different betagamma-responsive effectors.
- The C-terminal region of the G protein beta subunit is critical for PLC-beta2 activation.
- This finding provides insight into the differential signaling capabilities of the G protein betagamma complex.