G protein-effector coupling: interactions of recombinant inhibitory gamma subunit with transducin and phosphodiesterase.
A bacterial expression vector for the inhibitory gamma subunit of retinal rod phosphodiesterase has been constructed by inserting a mouse gamma cDNA into pUC19. Escherichia coli 222 transformed with this plasmid produces a 12-kDa recombinant protein consisting of 18 additional amino acids attached to the amino terminus of gamma. The fusion protein, designated beta-gal-gamma, has been refolded into an active form in formic acid and partially purified by gel filtration chromatography. Despite a large extended sequence at the amino terminus, beta-gal-gamma is able to inhibit the activity of trypsin-activated phosphodiesterase, bind tightly to the catalytic alpha beta subunits, and interact with the alpha subunit of transducin in a nucleotide-dependent manner. The availability of large quantities of active bacterial gamma, together with the ability to change its primary structure by site-directed mutagenesis, promises to provide considerable new information on the interaction between transducin and phosphodiesterase, as well as insights into the molecular mechanism of G protein-effector coupling.
Griswold-Prenner
I
I
Jules Stein Eye Institute, University of California School of Medicine, Los Angeles 90024.
Tuteja
N
N
Farber
D B
DB
Fung
B K
BK
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015293:Transducin / Q000378:metabolism*
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins; D015293:Transducin