Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein α subunit (Gα), thereby generating two potential signaling molecules, Gα-GTP and free Gβγ. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Ga mutation. Here we describe a mutant α subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the α subunit (αs) of Gs three separate mutations chosen because they impair αs function in complementary ways: the A366S mutant reduces affinity of αs for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active αs mutant (αs-R201C) or to agonists for receptors that activate Gq or Gi. This αs triple mutant and cognate Gα mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.

Original languageEnglish
Pages (from-to)499-504
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number2
StatePublished - Jan 19 1999


  • Adenylyl cyclase
  • Dominant negative
  • Trimeric G proteins


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