title: Studies on G-protein alpha.betagamma heterotrimer formation reveal a putative S-prenyl-binding site in the alpha subunit.
abstract: The alpha and betagamma subunits of heterotrimeric G-proteins contain specific lipid modifications, which are required for their biological function. However, the relevance of these modifications to the interactions within the heterotrimeric G-protein is not fully understood. In order to explore the role of the S-prenyl moiety of the isoprenylated betagamma dimer of retinal transducin, betagamma(t), in the formation of the heterotrimeric complex with the corresponding N-acylated alpha subunit, alpha(t), we employed purified fully processed subunits, which are soluble in aqueous solutions without detergents. Pertussis-toxin-mediated [(32)P]ADP-ribosylation of alpha(t) is strongly stimulated (approximately 10-fold) in the presence of betagamma(t) and can thus serve as a measure for heterotrimer formation. Using this assay, preincubation of alpha(t) with S-prenyl analogues containing farnesyl or geranylgeranyl moieties was found to inhibit heterotrimer formation in a dose-dependent manner. The inhibition was competitive and reversible, as indicated by its reversal upon increase of the betagamma(t) dimer concentration or by removal of the S-prenyl analogue using gel filtration. The competitive nature of the inhibition is supported by the marked attenuation of the inhibition when the S-prenyl analogue was added to alpha(t) together with or after betagamma(t). The inhibition does not involve interaction with the alpha(t) acyl group, since an S-prenyl analogue inhibited the [(32)P]ADP-ribosylation of an unlipidated alpha(t) mutant. These data suggest the existence of a hitherto unrecognized S-prenyl-binding site in alpha(t), which is critical for its interaction with prenylated betagamma(t).
authors.affiliation: Department of Pharmacology and Toxicology, University of Ulm, 89069 Ulm, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dietrich
authors.forename: Alexander
authors.initials: A
authors.lastname: Scheer
authors.forename: Daria
authors.initials: D
authors.lastname: Illenberger
authors.forename: Yoel
authors.initials: Y
authors.lastname: Kloog
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
authors.forename: Peter
authors.initials: P
authors.lastname: Gierschik
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D005204:Farnesol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D017368:Protein Prenylation; D012459:Salicylates / Q000378:metabolism / Q000494:pharmacology; D013729:Terpenes / Q000378:metabolism; D015293:Transducin / Q000378:metabolism
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D012459:Salicylates; D013729:Terpenes; C093323:farnesylthiosalicylic acid; D005204:Farnesol; D037342:Pertussis Toxin; D015293:Transducin; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine