The Binding of Activated Gα to Phospholipase C-β Exhibits Anomalous Affinity

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2017 Aug 27

PMID 28842497

Citations 6

Authors

Punya Navaratnarajah

Anne Gershenson

Elliott M Ross

Affiliations

Soon will be listed here.

Abstract

Upon activation by the G family of Gα subunits, Gβγ subunits, and some Rho family GTPases, phospholipase C-β (PLC-β) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-β isoforms also function as GTPase-activating proteins, potentiating G deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-β3 binding to Gα FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded values of about 200 nm for PLC-β3-Gα binding. This is 50-100 times greater than the EC for Gα-mediated PLC-β3 activation and for the Gα GTPase-activating protein activity of PLC-β. The measured was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca, or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a of 200 nm We determined that PLC-β3 hysteresis, whereby PLC-β3 remains active for some time following either Gα-PLC-β3 dissociation or PLC-β3-potentiated Gα deactivation, is not sufficient to explain the observed discrepancy between EC and These results indicate that the mechanism by which Gα and PLC-β3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.

Citing Articles

Systems modeling of oncogenic G-protein and GPCR signaling reveals unexpected differences in downstream pathway activation.

Trogdon M, Abbott K, Arang N, Lande K, Kaur N, Tong M NPJ Syst Biol Appl. 2024; 10(1):75.

PMID: 39013872 PMC: 11252164. DOI: 10.1038/s41540-024-00400-1.

A Model Reveals the Potential Role for in Retinal Disease.

Chen W, Zhong W, Yu L, Lin X, Xie J, Liu Z Int J Mol Sci. 2024; 25(2).

PMID: 38255972 PMC: 10815649. DOI: 10.3390/ijms25020899.

Dissociation of the G protein βγ from the Gq-PLCβ complex partially attenuates PIP2 hydrolysis.

Kankanamge D, Ubeysinghe S, Tennakoon M, Pantula P, Mitra K, Giri L J Biol Chem. 2021; 296:100702.

PMID: 33901492 PMC: 8138763. DOI: 10.1016/j.jbc.2021.100702.

DAPLE protein inhibits nucleotide exchange on Gα and Gα via the same motif that activates Gαi.

Marivin A, Maziarz M, Zhao J, DiGiacomo V, Olmos Calvo I, Mann E J Biol Chem. 2020; 295(8):2270-2284.

PMID: 31949046 PMC: 7039542. DOI: 10.1074/jbc.RA119.011648.

Phospholipase C.

Bill C, Vines C Adv Exp Med Biol. 2019; 1131:215-242.

PMID: 31646512 PMC: 7790445. DOI: 10.1007/978-3-030-12457-1_9.

References

Singer A, Waldo G, Harden T, Sondek J . A unique fold of phospholipase C-beta mediates dimerization and interaction with G alpha q. Nat Struct Biol. 2001; 9(1):32-6. DOI: 10.1038/nsb731. View

Mukhopadhyay S, Ross E . Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins. Proc Natl Acad Sci U S A. 1999; 96(17):9539-44. PMC: 22244. DOI: 10.1073/pnas.96.17.9539. View

Illenberger D, Walliser C, Nurnberg B, Diaz Lorente M, Gierschik P . Specificity and structural requirements of phospholipase C-beta stimulation by Rho GTPases versus G protein beta gamma dimers. J Biol Chem. 2002; 278(5):3006-14. DOI: 10.1074/jbc.M208282200. View

Kruse A, Hu J, Pan A, Arlow D, Rosenbaum D, Rosemond E . Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature. 2012; 482(7386):552-6. PMC: 3529910. DOI: 10.1038/nature10867. View

Lee C, Lee K, Lee S, Park D, Rhee S . Regulation of phospholipase C-beta 4 by ribonucleotides and the alpha subunit of Gq. J Biol Chem. 1994; 269(41):25335-8. View

Lyon A, Begley J, Manett T, Tesmer J . Molecular mechanisms of phospholipase C β3 autoinhibition. Structure. 2014; 22(12):1844-1854. PMC: 4308481. DOI: 10.1016/j.str.2014.10.008. View

Chan P, Gabay M, Wright F, Kan W, Oner S, Lanier S . Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf). J Biol Chem. 2010; 286(4):2625-35. PMC: 3024758. DOI: 10.1074/jbc.M110.178897. View

Lyon A, Tesmer V, Dhamsania V, Thal D, Gutierrez J, Chowdhury S . An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation. Nat Struct Mol Biol. 2011; 18(9):999-1005. PMC: 3168981. DOI: 10.1038/nsmb.2095. View

Kozasa T, GILMAN A . Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of alpha 12 and inhibition of adenylyl cyclase by alpha z. J Biol Chem. 1995; 270(4):1734-41. DOI: 10.1074/jbc.270.4.1734. View

10.

Chidiac P, Markin V, Ross E . Kinetic control of guanine nucleotide binding to soluble Galpha(q). Biochem Pharmacol. 1999; 58(1):39-48. DOI: 10.1016/s0006-2952(99)00080-5. View

11.

Hughes T, Zhang H, Logothetis D, Berlot C . Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by.... J Biol Chem. 2000; 276(6):4227-35. DOI: 10.1074/jbc.M007608200. View

12.

Gelb M, Jain M, Hanel A, Berg O . Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2. Annu Rev Biochem. 1995; 64:653-88. DOI: 10.1146/annurev.bi.64.070195.003253. View

13.

Hess S, Huang S, Heikal A, Webb W . Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry. 2002; 41(3):697-705. DOI: 10.1021/bi0118512. View

14.

Biddlecome G, Berstein G, Ross E . Regulation of phospholipase C-beta1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation. J Biol Chem. 1996; 271(14):7999-8007. DOI: 10.1074/jbc.271.14.7999. View

15.

Cheng J, Karri S, Grauffel C, Wang F, Reuter N, Roberts M . Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding?. Biophys J. 2013; 104(1):185-95. PMC: 3540254. DOI: 10.1016/j.bpj.2012.11.015. View

16.

Jensen J, Lyssand J, Hague C, Hille B . Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels. J Gen Physiol. 2009; 133(4):347-59. PMC: 2699104. DOI: 10.1085/jgp.200810075. View

17.

Jezyk M, Snyder J, Gershberg S, Worthylake D, Harden T, Sondek J . Crystal structure of Rac1 bound to its effector phospholipase C-beta2. Nat Struct Mol Biol. 2006; 13(12):1135-40. DOI: 10.1038/nsmb1175. View

18.

Padrick S, Brautigam C . Evaluating the stoichiometry of macromolecular complexes using multisignal sedimentation velocity. Methods. 2011; 54(1):39-55. PMC: 3147156. DOI: 10.1016/j.ymeth.2011.01.002. View

19.

Smith M, Liu Y, Matthews N, Rhee S, Sung W, Kung H . Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity. Proc Natl Acad Sci U S A. 1994; 91(14):6554-8. PMC: 44241. DOI: 10.1073/pnas.91.14.6554. View

20.

Kadamur G, Ross E . Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site. J Biol Chem. 2016; 291(21):11394-406. PMC: 4900283. DOI: 10.1074/jbc.M116.723940. View