Go is a member of a family of guanyl nucleotide binding proteins (GNPs) which are involved in the regulation of receptor- associated phenomena. Go has been shown to interact functionally with the photo-receptor rhodopsin and with muscarinic receptors. As has been observed with all other GNPs, Go exists as a heterotrimer of alpha, beta and gamma subunits. A cDNA clone, containing the complete protein coding sequence for the alpha subunit of bovine retina Go (Go alpha) has been isolated and sequenced. This clone has been utilized for expression in both prokaryotic and eukaryotic hosts. The cDNA, starting with the ATG initiation codon and comprising the entire coding region and approximately 200 bases of the 3' untranslated region was ligated into the EcoRl site of pRC-23, and E. coli expression vector. Upon induction, Go alpha is produced at a level approximating 1-2% of the total cellular protein. As demonstrated by western analysis, this recombinant protein (rGo alpha) is immunoreactive with polyclonal antisera. rGo alpha also serves as a substrate for ADP-ribosylation by pertussin toxin. Stimulation of pertussis toxin catalyzed ADP-ribosylation of rGo alpha is observed in the presence of the beta and gamma subunits of transducin. Gene transfer experiments have been carried out in which the Go alpha cDNA was transfected into COS cells, utilizing a vector with an SV40 promoter. Cytoplasmic dot-blot assays demonstrated that transcription occurred within 12 hours of uptake and continued for up to 80 hours.
