G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells
1996
Lindorfer, M.A. | Sherman, N.E. | Woodfork, K.A. | Fletcher, J.E. | Hunt, D.F. | Garrison, J.C.
The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the beta gamma dimer, modifications that are essential for alpha-beta gamma, beta gamma-receptor, and beta gamma-effector interactions. We have determined the specific prenyl group present on the beta 1 gamma 1, beta 1 gamma 2, and beta 1 gamma 3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma 1 and gamma 2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma 1 CaaX sequence (gamma 1-S74L) resulted in geranylgeranylation of the resulting gamma 1 subunit, and alteration of the gamma 2 CaaX sequence to CAIS (gamma 2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta 1, and the resulting beta gamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the beta gamma subunits participate.
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