Purification and characterization of G(oα) and three types of G(iα) after expression in Escherichia coli

Complementary DNAs for the G protein α subunits G(iα1), G(iα2), G(iα3), and G(oα) were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with βγ subunits. The rates of dissociation of GDP from G(iα1) and G(iα3) (0.03 min^-1) are an order of magnitude slower than that from rG(oα); release of GDP from G(iα2) is also relatively slow (0.07 min^-1). However, the values of k(cat) for the hydrolysis of GTP by rG(oα) and the three rG(iα) proteins are approximately the same, about 2 min^-1 at 20°C. The recombinant proteins restore inhibition of Ca²⁺ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin , while G(iα2) was inactive with neuropeptide Y. Despite these results, high concentrations of activated G(iα) proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or G(sα), the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein βγ subunits are primarily responsible for inhibition of adenylyl cyclase activity.