Activating and inactivating mutations of the α subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells

Sylvie Hermouet, John J. Merendino, J. Silvio Gutkind, Allen M. Spiegel

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Abstract

Previous studies have demonstrated that mutations of highly conserved residues in the α subunit of Gss) 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.

Original languageEnglish (US)
Pages (from-to)10455-10459
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number23
DOIs
Publication statusPublished - Jan 1 1991
Externally publishedYes

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Keywords

  • ADP-ribosylation
  • Guanine nucleotide-binding proteins
  • Site-directed mutagenesis
  • Stable transfection

ASJC Scopus subject areas

  • General

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