A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding.
The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.
Graf
R
R
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Mattera
R
R
Codina
J
J
Estes
M K
MK
Birnbaumer
L
L
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006882:Hydroxyapatites; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D016366:Open Reading Frames; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D037342:Pertussis Toxin; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006882:Hydroxyapatites; D046911:Macromolecular Substances; D009227:Myristic Acids; D010169:Palmitic Acids; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D017886:Durapatite; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins