Effects of overexpression of stimulatory G proteins on the expression of the other G proteins in COS-1 cells.
- Author:
So Yeon KIM
1
;
Ik Soon JANG
;
Jung Mee YANG
;
Sahng June KWAK
;
Yong Sung JUHNN
Author Information
1. SEOUL NATL UNIV, COLL MED, DEPT BIOCHEM, SEOUL 110799, SOUTH KOREA.
- Publication Type:Original Article
- Keywords:
adenosine cyclic monophosphate;
guanine nucleotide regulatory proteins;
recombinant proteins;
protein engineering;
Western blotting
- MeSH:
Animals;
Blotting, Western;
Carrier Proteins;
Colforsin;
COS Cells*;
Cyclic AMP;
DEAE-Dextran;
GTP-Binding Proteins*;
Isoproterenol;
Protein Engineering;
Recombinant Proteins;
Transfection
- From:Experimental & Molecular Medicine
1997;29(2):133-137
- CountryRepublic of Korea
- Language:English
-
Abstract:
To investigate the interaction of stimulatory GTP binding protein (G(s)) pathways with others, we overexpressed wild type alpha subunit of G(s) (G(s) alpha), constitutively activated R201E G(s) alpha, and dominant negative G226A G(s) alpha in COS-1 cells by transfection with DEAE-dextran, respectively, The expression of various G proteins in the transfected cells was analyzed after 72 h by quantitative Western blots, and cAMP production by stimulation with isoproterenol and forskolin was quantitated using cAMP binding proteins, The expression of Gs alpha increased about 5-fold in the transfected cells, with concomitant increase in the small forms. However, there was no significant alteration the in the level of the alpha subunit of inhibitory G protein (G(i)) and G(q), and the beta subunits of G proteins. The cAMP level without stimulation increased in the cells transfected with G(s) alpha regardless to the type of mutation, Treatment with either isoproterenol or forskolin resulted in comparable increase of the cAMP level in all the transfected cells, though the ratio to its respective basal level was smaller in the G(s) alpha-transfected cells, From this experiment, we found that the expression of the other G proteins and the signaling pathway producing cAMP did not change significantly by transiently expressing wild type, constitutively activated type, and dominant negative type of G(s) alpha. Analysis of the effects of long-term expression of Gs alpha would contribute to better understanding on how the G(s) alpha signaling system interacts with other signaling pathways and how it adapts to the changed environments.
