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.