Résumé
How receptors catalyze exchange of GTP for GDP bound to the Gα
subunit of trimeric G proteins is not known. One proposal is that the
receptor uses the G protein's βγ heterodimer as a lever, tilting
it to pull open the guanine nucleotide binding pocket of Gα. To test
this possibility, we designed a mutant Gα that would bind to βγ
in the tilted conformation. To do so, we excised a helical turn (four
residues) from the N-terminal region of αs, the α
subunit of GS, the stimulatory regulator of adenylyl
cyclase. In the presence, but not in the absence, of transiently
expressed β1 and γ2, this mutant
(αsΔ), markedly stimulated cAMP accumulation. This
effect depended on the ability of the coexpressed β protein to
interact normally with the lip of the nucleotide binding pocket of
αsΔ. We substituted alanine for an aspartate in
β1 that binds to a lysine (K206) in the lip of the α
subunit's nucleotide binding pocket. Coexpressed with
αsΔ and γ2, this mutant,
β1-D228A, elevated cAMP much less than did
β1-wild type; it did bind to αsΔ
normally, however, as indicated by its unimpaired ability to target
αsΔ to the plasma membrane. We conclude that βγ can
activate αs and that this effect probably involves both a
tilt of βγ relative to αs and interaction of β with
the lip of the nucleotide binding pocket. We speculate that receptors
use a similar mechanism to activate trimeric G proteins.