Year-end Sale % OFF 
Abstract
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.
Highlights
The ␣ and ␥ subunits of heterotrimeric G proteins1 undergo post-translational modification with lipids that are essential for their interaction with receptors and downstream effectors [1]
The results presented here help define ␥ subunit prenylation in the Sf9 cell system and provide the basis upon which to investigate the role prenylation plays in the interaction of the ␥ dimer with receptors and with effectors
These ␥ dimers were purified by ion exchange and ␣-agarose affinity chromatography, and all dimers shown are essentially free of contaminating proteins
Summary
The ␣ and ␥ subunits of heterotrimeric G proteins undergo post-translational modification with lipids that are essential for their interaction with receptors and downstream effectors [1]. The 11 known ␥ subunit isoforms all have CaaX motifs at their carboxyl terminus [2,3,4] which direct post-translational modifications including attachment of a prenyl group to the cysteine side chain, proteolytic cleavage of the aaX tripeptide, and methylation of the resulting carboxyl terminus [5]. We report here the expression of these constructs in the baculovirus Sf9 cell system and purification to homogeneity of the wild type and altered ␥ subunits complexed with the 1 subunit. We have verified using mass spectrometric techniques that wild type and altered CaaX sequences result in the expected post-translational modifications in the baculovirus Sf9 cell expression system. The accompanying report describes the effect of these prenyl modifications on the ability of the heterotrimeric G protein to interact with the A1 adenosine receptor [19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
