The long term goal is to use molecular genetic methods to understand the role of G protein diversity in mammalian signal transduction. While the a subunits are a large family of proteins that are known to modulate effector function, the role of the beta and gamma subunits is unclear.
The specific aims will be to determine the structural diversity among these subunits and the relationship between the structure and function. Molecular cloning techniques that identify rare or transiently expressed beta or gamma subunits will be used to identify novel cDNAs in rat or mouse. The screening will be specifically directed at identifying subtypes expressed in the nervous system. The expression of RNA for novel subunits will be examined in mouse tissues. Antibodies specific to each subunit type will be generated. These antibodies will be used to examine protein expression in different situations. One will be an assay to determine if specific associations of beta and gamma subtypes take place. This assay will involve the expression of cDNAs for beta and gamma subunits in PC12 cells and examining cytosolic and membrane proteins with the antibodies. There is recent evidence that coexpression of beta and gamma subunits is essential to stabilize these proteins. Mutant and chimeric molecules will be used to determine the regions required for interaction of beta and gamma subunits. Mutants that affect the ability of the beta-gamma complex to associate with the cell membrane will be introduced stably into PC12 cells. Such mutants have the potential to prevent G protein activation in a signalling pathway and can therefore be dominant and negative. A signalling network responds to muscarine and bradykinin in PC12 cells. These pathways will be assayed in stable transfectants carrying mutants. Human disease states can arise from the constitutive activation of signalling pathways due to mutations in the receptor or G protein a subunits. Dominant negative mutants of beta or gamma subunits could potentially block such pathways.
| Azpiazu, I; Gautam, N (2006) A G protein gamma subunit peptide stabilizes a novel muscarinic receptor state. Biochem Biophys Res Commun 341:904-10 |
| Kisselev, O G; Ermolaeva, M V; Gautam, N (1994) A farnesylated domain in the G protein gamma subunit is a specific determinant of receptor coupling. J Biol Chem 269:21399-402 |
| Pronin, A N; Gautam, N (1993) Proper processing of a G protein gamma subunit depends on complex formation with a beta subunit. FEBS Lett 328:89-93 |
| Kisselev, O; Gautam, N (1993) Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein. J Biol Chem 268:24519-22 |
