S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) The Seoul Journal of Medicine The Seoul Journal of Medicine Vol. 34 No.4 (1993)
Influences of Nonmyristoylated alpha Subunit of Gi2 on Adenylate Cyclase Signaling Pathway in COS-7 Cells
- Juhnn, Yong-Sung
- Issue Date
- Seoul J Med, Vol.34 No.4, pp. 245-251
- 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.