Abstract
We have previously shown that isoprenylation and/or additional post-translational processing of the G protein γ1 subunit carboxyl terminus is required for β1γ1 subunit stimulation of phospholipase C-β2 (PLCβ2) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171−178]. To examine whether isoprenylation of the γ1 subunit alone is sufficient for β1γ1-mediated PLCβ2 stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant β1γ1 dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the β1γ1 dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the γ1 subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated β1γ1 dimers with a recombinant PLCβ2 isozyme revealed that farnesylation rendered recombinant nonisoprenylated β1γ1 dimers capable of stimulating PLCβ2 and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated β1γ1 dimers than for fully modified native β1γ1 purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the γ subunit is both necessary and sufficient for βγ dimer-mediated stimulation of phospholipase C.
Original language | English |
---|---|
Pages (from-to) | 15174-15182 |
Number of pages | 9 |
Journal | Biochemistry |
Volume | 35 |
Issue number | 48 |
DOIs | |
Publication status | Published - 03 Dec 1996 |
Externally published | Yes |