G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells.
The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 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 gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma 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 betagamma subunits participate.
Lindorfer
M A
MA
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Sherman
N E
NE
Woodfork
K A
KA
Fletcher
J E
JE
Hunt
D F
DF
Garrison
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D011994:Recombinant Proteins; D019204:GTP-Binding Proteins