Mutation of Cysteine 214 in Gi1 Alpha Subunit Abolishes Its Endogenous GTPase Activity

Overview

Journal Biochem J

Specialty Biochemistry

Date 2004 Jan 17

PMID 14725508

Citations 1

Authors

Yuren Wang

Gregory Tawa

Deborah Smith

Girija Krishnamurthy

Kathleen H Young

Affiliations

Soon will be listed here.

Abstract

The functional consequences of the mutation of a conserved Cys-214 in Galpha(i1) have been investigated. We reported herein that substitutions of Cys-214 of Galpha(i1) to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [32P]GTP-bound Galpha(i1) C214A or [32P]GTP-bound Galpha(i1) C214W was at least 30-fold lower than that of the wild-type Galpha(i1) in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with betagamma subunits. Moreover, RGS4 protein, a GTPase-activating protein for Galpha(i1), cannot interact with Cys-214 mutants even in the presence of AlF4-, which induces the transition state of Galpha. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Galpha(i1) in cells.

Citing Articles

Dynamics of receptor/G protein coupling in living cells.

Hein P, Frank M, Hoffmann C, Lohse M, Bunemann M EMBO J. 2005; 24(23):4106-14.

PMID: 16292347 PMC: 1356310. DOI: 10.1038/sj.emboj.7600870.

References

Coleman D, Berghuis A, Lee E, Linder M, GILMAN A, Sprang S . Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science. 1994; 265(5177):1405-12. DOI: 10.1126/science.8073283. View

Coleman D, Lee E, Mixon M, Linder M, Berghuis A, GILMAN A . Crystallization and preliminary crystallographic studies of Gi alpha 1 and mutants of Gi alpha 1 in the GTP and GDP-bound states. J Mol Biol. 1994; 238(4):630-4. DOI: 10.1006/jmbi.1994.1320. View

Kleuss C, Raw A, Lee E, Sprang S, GILMAN A . Mechanism of GTP hydrolysis by G-protein alpha subunits. Proc Natl Acad Sci U S A. 1994; 91(21):9828-31. PMC: 44910. DOI: 10.1073/pnas.91.21.9828. View

Mixon M, Lee E, Coleman D, Berghuis A, GILMAN A, Sprang S . Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis. Science. 1995; 270(5238):954-60. DOI: 10.1126/science.270.5238.954. View

Garcia-Higuera I, Thomas T, Yi F, Neer E . Intersubunit surfaces in G protein alpha beta gamma heterotrimers. Analysis by cross-linking and mutagenesis of beta gamma. J Biol Chem. 1996; 271(1):528-35. DOI: 10.1074/jbc.271.1.528. View

Lambright D, Sondek J, Bohm A, Skiba N, Hamm H, SIGLER P . The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996; 379(6563):311-9. DOI: 10.1038/379311a0. View

Berman D, Wilkie T, GILMAN A . GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell. 1996; 86(3):445-52. DOI: 10.1016/s0092-8674(00)80117-8. View

Berman D, Kozasa T, GILMAN A . The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J Biol Chem. 1996; 271(44):27209-12. DOI: 10.1074/jbc.271.44.27209. View

Mejillano M, Shivanna B, Himes R . Studies on the nocodazole-induced GTPase activity of tubulin. Arch Biochem Biophys. 1996; 336(1):130-8. DOI: 10.1006/abbi.1996.0540. View

10.

Sprang S . G protein mechanisms: insights from structural analysis. Annu Rev Biochem. 1997; 66:639-78. DOI: 10.1146/annurev.biochem.66.1.639. View

11.

Raw A, Coleman D, GILMAN A, Sprang S . Structural and biochemical characterization of the GTPgammaS-, GDP.Pi-, and GDP-bound forms of a GTPase-deficient Gly42 --> Val mutant of Gialpha1. Biochemistry. 1998; 36(50):15660-9. DOI: 10.1021/bi971912p. View

12.

Lan K, Sarvazyan N, Taussig R, MacKenzie R, DiBello P, Dohlman H . A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins. J Biol Chem. 1998; 273(21):12794-7. DOI: 10.1074/jbc.273.21.12794. View

13.

Posner B, Mixon M, Wall M, Sprang S, GILMAN A . The A326S mutant of Gialpha1 as an approximation of the receptor-bound state. J Biol Chem. 1998; 273(34):21752-8. DOI: 10.1074/jbc.273.34.21752. View

14.

Apanovitch D, Iiri T, Karasawa T, Bourne H, Dohlman H . Second site suppressor mutations of a GTPase-deficient G-protein alpha-subunit. Selective inhibition of Gbeta gamma-mediated signaling. J Biol Chem. 1998; 273(44):28597-602. DOI: 10.1074/jbc.273.44.28597. View

15.

Coleman D, Sprang S . Structure of Gialpha1.GppNHp, autoinhibition in a galpha protein-substrate complex. J Biol Chem. 1999; 274(24):16669-72. DOI: 10.1074/jbc.274.24.16669. View

16.

Wang Y, Windh R, Chen C, Manning D . N-Myristoylation and betagamma play roles beyond anchorage in the palmitoylation of the G protein alpha(o) subunit. J Biol Chem. 1999; 274(52):37435-42. DOI: 10.1074/jbc.274.52.37435. View

17.

Wang Y, Ho G, Zhang J, Nieuwenhuijsen B, Edris W, Chanda P . Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling. J Biol Chem. 2002; 277(50):48325-32. DOI: 10.1074/jbc.M206116200. View

18.

Katada T, Ui M . Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982; 79(10):3129-33. PMC: 346367. DOI: 10.1073/pnas.79.10.3129. View

19.

Fung B, Nash C . Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin. J Biol Chem. 1983; 258(17):10503-10. View

20.

Van Dop C, Tsubokawa M, Bourne H, Ramachandran J . Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. J Biol Chem. 1984; 259(2):696-8. View