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- Title
Catalytic site mutations confer multiple states of G protein activation.
- Authors
Hewitt, Natalie; Ma, Ning; Arang, Nadia; Martin, Sarah A.; Prakash, Ajit; DiBerto, Jeffrey F.; Knight, Kevin M.; Ghosh, Soumadwip; Olsen, Reid H. J.; Roth, Bryan L.; Gutkind, J. Silvio; Vaidehi, Nagarajan; Campbell, Sharon L.; Dohlman, Henrik G.
- Abstract
Heterotrimeric guanine nucleotide–binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gαq or Gα11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein–coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance–based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands. The active states of Gα proteins: Ligand binding to a GPCR leads to G protein activation through the displacement of GDP by GTP at the α-subunit and the subsequent dissociation of Gα from Gβγ. The GTPase activity of Gα, which depends on a conserved glutamine residue, hydrolyzes GTP to GDP, thereby deactivating the G protein. Noting that rare mutations of the catalytic glutamine residue confer constitutive activity to some G proteins, Hewitt et al. analyzed the effect of mutating this residue to all other possible residues in multiple G protein α-subunits. Through biochemical, functional, and structural studies, the authors showed that whereas all possible mutants tested exhibited no GTPase activity, not all were constitutively active, some retained the ability to be further activated or inhibited by GPCRs, and some still bound to Gβγ. In addition, different mutations resulted in the generation of multiple active state conformations. These data suggest that the role of this conserved glutamine residue goes beyond catalysis to regulate G protein structure and function. —JFF
- Subjects
G protein coupled receptors; G proteins; MOLECULAR dynamics; LIGAND binding (Biochemistry); ALLOSTERIC regulation; MOLECULAR switches
- Publication
Science Signaling, 2023, Vol 16, Issue 772, p1
- ISSN
1945-0877
- Publication type
Article
- DOI
10.1126/scisignal.abq7842