G protein-effector coupling: interactions of recombinant inhibitory .gamma. subunit with transducin and phosphodiesterase

Abstract

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.