Abstract
We have determined the relative abilities of several members of the G protein beta and gamma subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian beta1 and gamma3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of beta and gamma subunit types varies widely. Third, the reporter activity evoked by a particular combination of beta and gamma subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid betagamma complex formed in the cell as determined by immunoprecipitation. These results suggest that different beta and gamma subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all gamma subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins.
Highlights
Most of the neurohormonal signaling pathways in mammals are mediated by heterotrimeric G proteins [1, 2]
In an assay of interaction between p53 and T antigen, it was shown that p53 mutants, whose interaction with T antigen could not be detected in an immunoprecipitation assay, were capable of interaction with T antigen since co-expression of the mutant p53 and T antigens as hybrids resulted in measurable reporter activity in the two-hybrid system [6]
The same study showed that in the case of mutant p53 proteins, whose interaction with T antigen could be detected by immunoprecipitation, there was a direct relationship between the amount of protein complex immunoprecipitated and the reporter activity detected when the same proteins were co-expressed in the two-hybrid system
Summary
Most of the neurohormonal signaling pathways in mammals are mediated by heterotrimeric G proteins [1, 2]. If the proteins interact with each other, the transcription factor domains are in proximity and capable of activating a promoter that controls reporter activity [6] We chose this system because it measures protein-protein interaction in a yeast cell and is a reasonably accurate reflection of the ability of the interacting proteins to form a complex in a cell. If ␥ complexes made up of different  and ␥ subunit types had different properties at each of these two points in a G protein-mediated signaling pathway, the mechanisms that control the formation of specific forms of the ␥ complex in a cell will regulate the signaling properties of a cell. The ability to assay interactions between two families of proteins with diverse structures allows us to examine the molecular basis of interactions between protein families that form a multimeric complex
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