Mutant G protein alpha subunit activated by Gbeta gamma: a model for receptor activation?
How receptors catalyze exchange of GTP for GDP bound to the Galpha subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's betagamma heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Galpha. To test this possibility, we designed a mutant Galpha that would bind to betagamma in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of alpha(s), the alpha subunit of G(S), the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed beta(1) and gamma(2), this mutant (alpha(s)Delta), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed beta protein to interact normally with the lip of the nucleotide binding pocket of alpha(s)Delta. We substituted alanine for an aspartate in beta(1) that binds to a lysine (K206) in the lip of the alpha subunit's nucleotide binding pocket. Coexpressed with alpha(s)Delta and gamma(2), this mutant, beta(1)-D228A, elevated cAMP much less than did beta(1)-wild type; it did bind to alpha(s)Delta normally, however, as indicated by its unimpaired ability to target alpha(s)Delta to the plasma membrane. We conclude that betagamma can activate alpha(s) and that this effect probably involves both a tilt of betagamma relative to alpha(s) and interaction of beta with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.
Rondard
P
P
Departments of Cellular and Molecular Pharmacology, Medicine, and the Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0450, USA.
Iiri
T
T
Srinivasan
S
S
Meng
E
E
Fujita
T
T
Bourne
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis
D006153:Guanosine Diphosphate; D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins