Open AccessStimulation of phospholipase C‐β 2 by recombinant guanine‐nucleotide‐binding protein βγ dimers produced in a baculovirus/insect cell expression system
Author(s) -
DIETRICH Alexander,
MEISTER Michael,
BRAZIL Derek,
CAMPS Montserrat,
GIERSCHIK Peter
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb19927.x
Subject(s) - gamma subunit , beta (programming language) , recombinant dna , g alpha subunit , mutant , phospholipase c , protein subunit , biochemistry , biology , microbiology and biotechnology , chemistry , signal transduction , gene , computer science , programming language
Recombinant wild‐type β 1 γ 1 dimers of signal‐transducing guanine nucleotide‐binding proteins (G proteins) and β 1 γ 1 dimers carrying a mutation known to block γ‐subunit isoprenylation (β 1 γ 1 C71S) were expressed in baculovirus‐infected insect cells. Both wild‐type and mutant β 1 γ 1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic‐radiolabeling studies revealed that the soluble β 1 γ 1 preparation contained approximately equal amounts of non‐isoprenylated and isoprenylated β 1 γ 1 dimers. Soluble wild‐type and mutant β 1 γ 1 dimers and native β 1 γ 1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C‐β 2 . Only isoprenylated β 1 γ 1 dimers were capable of stimulating phospholipase C‐β 2 . 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.