Stimulation of phospholipase C-beta 2 by recombinant guanine-nucleotide-binding protein beta gamma dimers produced in a baculovirus/insect cell expression system. Requirement of gamma-subunit isoprenylation for stimulation of phospholipase C.
Recombinant wild-type beta 1 gamma 1 dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta 1 gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta 1 gamma 1 C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta 1 gamma 1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta 1 gamma 1 preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta 1 gamma 1 dimers. Soluble wild-type and mutant beta 1 gamma 1 dimers and native beta 1 gamma 1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta 2. Only isoprenylated beta 1 gamma 1 dimers were capable of stimulating phospholipase C-beta 2. The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.
Dietrich
A
A
Molecular Pharmacology Division, German Cancer Research Center, Heidelberg.
Meister
M
M
Brazil
D
D
Camps
M
M
Gierschik
P
P
D000818:Animals; D016367:Baculoviridae; D002417:Cattle; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D046911:Macromolecular Substances; D009036:Moths; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012160:Retina / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins