Synthesis and characterization of fluorescently labeled bovine brain G protein subunits.
G proteins play an important role in transmitting hormonal signals, and fluorescence techniques would be useful to study their cellular distribution and mechanisms. To prepare active fluorescent G protein Go/Gi or beta gamma subunits were reacted with fluorescein isothiocyanate (FITC) to label the alpha (F-alpha) and gamma (F-gamma/beta) subunits or with (iodoacetamido)tetramethylrhodamine (TMR-IAA) to label the beta subunit (TMR-beta gamma). Unreacted dye was removed from the labeled proteins by ultrafiltration, followed by further purification using HPLC gel filtration. The molar ratios of dye to protein were 0.96 +/- 0.15, 0.59 +/- 0.07, and 1.37 +/- 0.09 for labeled alpha,beta, and gamma subunits, respectively. GTP gamma S binding to F-alpha and ADP-ribosylation by pertussis toxin of F-alpha were reduced to 63% and 78% of control, respectively. F-alpha was a heterogeneous population of alpha subunits. Active F-alpha containing less than one (0.7) label/subunit (F-alpha-Mono Q) was separated from unlabeled and multiply labeled F-alpha by Mono Q anion-exchange chromatography. F-alpha-Mono Q displayed reduced GTPase activity (turnover number was 46% of control), while GTP gamma S binding and ADP-ribosylation by pertussis toxin were only decreased to 78% and 82% of control, respectively. TMR-beta gamma and F-gamma/beta retain full function compared to native beta gamma, as measured by three methods: (1) TMR-beta gamma and F-gamma/beta are able to form heterotrimers with alpha o subunits, (2) TMR-beta gamma and F-gamma/beta support the ADP ribosylation of alpha o subunits by pertussis toxin, and (3) TMR-beta gamma and F-gamma/beta inhibit forskolin-stimulated adenylyl cyclase activity. The fluorescent G protein subunits will be valuable tools to study G protein mechanisms in reconstituted membranes and intact cells.
Kwon
G
G
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
Remmers
A E
AE
Datta
S
S
Neubig
R R
RR
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D012235:Rhodamines; D014357:Trypsin
D005456:Fluorescent Dyes; D012235:Rhodamines; C034996:tetramethylrhodamine iodoacetamide; D014357:Trypsin; D019204:GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate