title: Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.
abstract: We have previously shown that isoprenylation and/or additional post-translational processing of the G protein gamma 1 subunit carboxyl terminus is required for beta 1 gamma 1 subunit stimulation of phospholipase C-beta 2 (PLC beta 2) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma 1 subunit alone is sufficient for beta 1 gamma 1-mediated PLC beta 2 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 beta 1 gamma 1 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 beta 1 gamma 1 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 gamma 1 subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta 1 gamma 1 dimers with a recombinant PLC beta 2 isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta 1 gamma 1 dimers capable of stimulating PLC beta 2 and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta 1 gamma 1 dimers than for fully modified native beta 1 gamma 1 purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.
authors.affiliation: Department of Pharmacology and toxicology, University of Ulm, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Dietrich
authors.forename: D
authors.initials: D
authors.lastname: Brazil
authors.forename: O N
authors.initials: ON
authors.lastname: Jensen
authors.forename: M
authors.initials: M
authors.lastname: Meister
authors.forename: M
authors.initials: M
authors.lastname: Schrader
authors.forename: J F
authors.initials: JF
authors.lastname: Moomaw
authors.forename: M
authors.initials: M
authors.lastname: Mann
authors.forename: D
authors.initials: D
authors.lastname: Illenberger
authors.forename: P
authors.initials: P
authors.lastname: Gierschik
mesh_terms: D019883:Alkyl and Aryl Transferases; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D051231:Farnesyltranstransferase; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011487:Protein Conformation; D017368:Protein Prenylation; D012717:Sesquiterpenes; D014166:Transferases / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D010738:Type C Phospholipases; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D019204:GTP-Binding Proteins