Influences of Nonmyristoylated alpha Subunit of Gi2 on Adenylate Cyclase Signaling Pathway in COS-7 Cells

Abstract

To investigate the functional significance of myristoylation of inhibitory GlP binding protein IY. subunit, the rat eDNA encoding GiZIY. was mutated at the glycine-2 modified normally by myristoylation. The glycine was replaced with alanine or deleted to construct G2A GiZiY. and (~2-9)GzIY. mutants, respectively. The mutant and wild type GiZiY. cDNAs were expressed transiently in COS-7 cells, and the intracellular localization of the proteins were analyzed by SDS-PAGE and immunoblot. Wild type Gi2IY. protein was localized mainly on the particulate fraction, but the nonmyristoylated G2A- and (~2-9)GiZIY. proteins were localized to the cytosol. These results confirmed that myristoylation of GzIY. protein is required for its membrane binding as well as for GillY. and GOiY.. The basal level of cAMP in the COS cells transfected with Gz·iY. cDNAs ranged from 5.8 to 31 pmol/mq-proteins. When cells were treated with 10 liM isoproterenoi, the cAMP level increased by 8- to 20 fold from the basal state, but the levels in COS cells expressing nonmyristoylated mutants were lower than that of control. Forskolin-stimulated accumulation of cAMP was increased in all the cells, and it also decreased in cells expressing nonmyristoylated GizCi by 15% to 45% from the control. There was no significant alteration in the immunoreactivity of GsiY. quantitated from the immunoblot. These results suggested that the nonmyristoylated GzIY. may decrease the adenylate cyclase activity. It might be possible that there is some crosstalk between the expression of Gu« and that of adenylate cyclase.