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Mutant G protein alpha subunit activated by Gbeta gamma: a model for receptor activation?
P Rondard1, T Iiri, S Srinivasan
1Departments of Cellular and Molecular Pharmacology, Medicine, and the Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0450, USA.
Summary
G protein betagamma subunits activate Galpha by tilting and interacting with its nucleotide pocket. This mechanism, involving G protein signaling, may be how receptors activate G proteins.
Area of Science:
- Molecular Biology
- Cell Signaling
Background:
- Receptor-mediated activation of trimeric G proteins involves the exchange of GTP for GDP on the Galpha subunit.
- The precise mechanism by which receptors catalyze this nucleotide exchange is not fully understood.
Purpose of the Study:
- To investigate the role of the G protein betagamma heterodimer in catalyzing GTP/GDP exchange on Galpha.
- To test the hypothesis that betagamma acts as a lever to open the Galpha nucleotide binding pocket.
Main Methods:
- A mutant Galpha subunit (alpha(s)Delta) was engineered by deleting four residues from its N-terminal region to favor binding to betagamma in a tilted conformation.
- The functional interaction between the mutant Galpha and beta/gamma subunits was assessed by measuring cAMP accumulation.
- Mutagenesis of a specific aspartate residue in beta(1) (beta(1)-D228A) was performed to disrupt its interaction with the Galpha nucleotide pocket lip.
Main Results:
- The alpha(s)Delta mutant significantly stimulated cAMP accumulation in the presence of beta(1) and gamma(2) subunits, but not in their absence.
- This stimulation was dependent on the interaction between beta(1) and the lip of the alpha(s)Delta nucleotide binding pocket.
- The beta(1)-D228A mutant showed reduced stimulation of cAMP, despite normal binding to alpha(s)Delta and targeting to the plasma membrane.
Conclusions:
- The G protein betagamma subunit can directly activate the Galpha subunit.
- This activation likely involves a conformational change in betagamma (tilt) relative to Galpha and direct interaction of beta with the Galpha nucleotide binding pocket lip.
- This mechanism may be conserved in receptor-mediated activation of trimeric G proteins.
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