Ribozyme-mediated suppression of the G protein gamma(7) subunit suggests a role in hormone regulation of adenylylcyclase activity

Human HEK 293 cells present a simple and tractable system to directly test the hypothesis that the G protein gamma subunits contribute to the specificity of receptor signaling pathways in vivo. To begin to elucidate the functions of the individual gamma subunits in these cells, a ribozyme strategy was used to specifically inactivate the mRNA encoding the gamma(7) subunit. A phosphorothioated DNA-RNA chimeric hammerhead ribozyme was constructed and analyzed for specificity toward the targeted gamma(7) subunit. In vitro cleavage analysis of this ribozyme revealed a highly efficient cleavage activity directed exclusively toward the gamma(7) RNA transcript. In particular, this ribozyme did not result in cleavage of the gamma(12) RNA transcript, which is 75% identical to the gamma(7) RNA transcript. Using a transient transfection assay, in vivo analysis of this ribozyme showed a specific reduction in both the mRNA and protein expression of the gamma(7) subunit in HEK 293 cells. Coincident with this loss in gamma(7) subunit, there was a specific reduction in the protein expression of the beta(1) subunit, suggesting that the beta(1) and gamma(7) subunits may functionally interact to form a beta gamma dimer in vivo. Functional analysis of the consequences of ribozyme-mediated suppression of the gamma(7) subunit expression indicated that it was associated with significant attenuation of isoproterenol-, but not prostaglandin E-1-, stimulated adenylylcyclase activity. Suppression of the gamma(7) subunit expression had no effect on carbachol- and ATP-mediated stimulation of phosphatidylinositol turnover. Taken together, these results not only indicate the feasibility of using the ribozyme technology to determine the roles of individual gamma subunits in receptor-G protein-effector pathways in vivo, but they point to a specific role of the gamma(7) subunit in the regulation of adenylylcyclase activity in response to isoproterenol.