Stimulation of phospholipase C-β2 by recombinant guanine-nucleotide-binding protein βγ dimers produced in a baculovirus/insect cell expression system: requirement of γ-subunit isoprenylation for stimulation of phospholipase C

Recombinant wild-type β1γ1 dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and β1γ1 dimers carrying a mutation known to block γ-subunit isoprenylation (β1γ1C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant β1γ1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble β1γ1 preparation contained approximately equal amounts of non-isoprenylated and isoprenylated β1γ1 dimers. Soluble wild-type and mutant β1γ1 dimers and native β1γ1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C-β2. Only isoprenylated β1γ1 dimers were capable of stimulating phospholipase C-β2. The results show that γ-subunit isoprenylation and/or additional post-translational processing of the protein are required for βγ subunit stimulation of phospholipase C.