TY - JOUR
T1 - Activating and inactivating mutations of the α subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells
AU - Hermouet, Sylvie
AU - Merendino, John J.
AU - Silvio Gutkind, J.
AU - Spiegel, Allen M.
PY - 1991
Y1 - 1991
N2 - Previous studies have demonstrated that mutations of highly conserved residues in the α subunit of Gs (αs) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding Gi2 α subunit (αi2 containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type αi2, Q205L αi2, and G204A αi2 was confirmed in transfected cells by immunoblot analysis. The overexpression of all three αi2 proteins was accompanied by an increase in β-subunit expression. Q205L αi2 was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type βi2. Expression of Q205L βi2 markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A βi2 increased intracellular cAMP and was resistant to guanosine 5′-[γ-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive αi2. Transfection of wild-type, Q205L, or G204A αi2 cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L αi2 induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in {3H]thymidine incorporation. Q205L αi2 cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A αi2 slowed the growth of NIH 3T3 cells. We conclude that αi2 plays an important role in regulation of fibroblast growth.
AB - Previous studies have demonstrated that mutations of highly conserved residues in the α subunit of Gs (αs) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding Gi2 α subunit (αi2 containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type αi2, Q205L αi2, and G204A αi2 was confirmed in transfected cells by immunoblot analysis. The overexpression of all three αi2 proteins was accompanied by an increase in β-subunit expression. Q205L αi2 was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type βi2. Expression of Q205L βi2 markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A βi2 increased intracellular cAMP and was resistant to guanosine 5′-[γ-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive αi2. Transfection of wild-type, Q205L, or G204A αi2 cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L αi2 induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in {3H]thymidine incorporation. Q205L αi2 cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A αi2 slowed the growth of NIH 3T3 cells. We conclude that αi2 plays an important role in regulation of fibroblast growth.
KW - ADP-ribosylation
KW - Guanine nucleotide-binding proteins
KW - Site-directed mutagenesis
KW - Stable transfection
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U2 - 10.1073/pnas.88.23.10455
DO - 10.1073/pnas.88.23.10455
M3 - Article
C2 - 1660138
AN - SCOPUS:0025880896
SN - 0027-8424
VL - 88
SP - 10455
EP - 10459
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 23
ER -