Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gβ and Gγ Subunits in C57Bl6/J Brain Synaptosomes

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2017 Sep 8

PMID 28880079

Citations 8

Authors

Yun Young Yim

W Hayes McDonald

Karren Hyde

Osvaldo Cruz-Rodriguez

John J G Tesmer

Heidi E Hamm

Affiliations

Soon will be listed here.

Abstract

Gβγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gβ and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gβ and Gγ subunits selectively interact to form unique Gβγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gβγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gβ and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gβ and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gβ and Gγ subunits and their subcellular localizations. For example, Gβ was mostly localized at the membrane while Gβ was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gβ and Gγ subunits may affect the Gβγ dimerization and Gβγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gβ and Gγ subunits but also new insights into the in vivo distribution of Gβ and Gγ subunits, and Gβγ dimer assembly in normal brain function.

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References

Pronin A, Gautam N . Interaction between G-protein beta and gamma subunit types is selective. Proc Natl Acad Sci U S A. 1992; 89(13):6220-4. PMC: 49470. DOI: 10.1073/pnas.89.13.6220. View

Fredriksson R, Lagerstrom M, Lundin L, Schioth H . The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol. 2003; 63(6):1256-72. DOI: 10.1124/mol.63.6.1256. View

Yasuda H, Lindorfer M, Myung C, Garrison J . Phosphorylation of the G protein gamma12 subunit regulates effector specificity. J Biol Chem. 1998; 273(34):21958-65. DOI: 10.1074/jbc.273.34.21958. View

Betke K, L Rose K, Friedman D, Baucum 2nd A, Hyde K, Schey K . Differential localization of G protein βγ subunits. Biochemistry. 2014; 53(14):2329-43. PMC: 4004276. DOI: 10.1021/bi500091p. View

Herlitze S, Garcia D, Mackie K, Hille B, Scheuer T, Catterall W . Modulation of Ca2+ channels by G-protein beta gamma subunits. Nature. 1996; 380(6571):258-62. DOI: 10.1038/380258a0. View

Currie K . Inhibition of Ca2+ channels and adrenal catecholamine release by G protein coupled receptors. Cell Mol Neurobiol. 2010; 30(8):1201-8. PMC: 3028936. DOI: 10.1007/s10571-010-9596-7. View

Hoofnagle A, Whiteaker J, Carr S, Kuhn E, Liu T, Massoni S . Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays. Clin Chem. 2016; 62(1):48-69. PMC: 4830481. DOI: 10.1373/clinchem.2015.250563. View

Cali J, Balcueva E, Rybalkin I, Robishaw J . Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning. J Biol Chem. 1992; 267(33):24023-7. View

Anderson G, Cao Y, Davidson S, Truong H, Pravetoni M, Thomas M . R7BP complexes with RGS9-2 and RGS7 in the striatum differentially control motor learning and locomotor responses to cocaine. Neuropsychopharmacology. 2010; 35(4):1040-50. PMC: 2887292. DOI: 10.1038/npp.2009.212. View

10.

Blackmer T, Larsen E, Bartleson C, Kowalchyk J, Yoon E, Preininger A . G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nat Neurosci. 2005; 8(4):421-5. DOI: 10.1038/nn1423. View

11.

Albert P, Robillard L . G protein specificity: traffic direction required. Cell Signal. 2002; 14(5):407-18. DOI: 10.1016/s0898-6568(01)00259-5. View

12.

Khan S, Sleno R, Gora S, Zylbergold P, Laverdure J, Labbe J . The expanding roles of Gβγ subunits in G protein-coupled receptor signaling and drug action. Pharmacol Rev. 2013; 65(2):545-77. DOI: 10.1124/pr.111.005603. View

13.

Chen C, Eversole-Cire P, Zhang H, Mancino V, Chen Y, He W . Instability of GGL domain-containing RGS proteins in mice lacking the G protein beta-subunit Gbeta5. Proc Natl Acad Sci U S A. 2003; 100(11):6604-9. PMC: 164494. DOI: 10.1073/pnas.0631825100. View

14.

Gautam N, Downes G, Yan K, Kisselev O . The G-protein betagamma complex. Cell Signal. 1998; 10(7):447-55. DOI: 10.1016/s0898-6568(98)00006-0. View

15.

Liang J, Cockett M, Khawaja X . Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain. J Neurochem. 1998; 71(1):345-55. View

16.

Dingus J, Wells C, Campbell L, Cleator J, Robinson K, Hildebrandt J . G Protein betagamma dimer formation: Gbeta and Ggamma differentially determine efficiency of in vitro dimer formation. Biochemistry. 2005; 44(35):11882-90. DOI: 10.1021/bi0504254. View

17.

Downes G, Gautam N . The G protein subunit gene families. Genomics. 2000; 62(3):544-52. DOI: 10.1006/geno.1999.5992. View

18.

Morishita R, Ueda H, Kato K, Asano T . Identification of two forms of the gamma subunit of G protein, gamma10 and gamma11, in bovine lung and their tissue distribution in the rat. FEBS Lett. 1998; 428(1-2):85-8. DOI: 10.1016/s0014-5793(98)00498-0. View

19.

Waldschmidt H, Homan K, Cruz-Rodriguez O, Cato M, Waninger-Saroni J, Larimore K . Structure-Based Design, Synthesis, and Biological Evaluation of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors. J Med Chem. 2016; 59(8):3793-807. PMC: 4890168. DOI: 10.1021/acs.jmedchem.5b02000. View

20.

Robishaw J, Berlot C . Translating G protein subunit diversity into functional specificity. Curr Opin Cell Biol. 2004; 16(2):206-9. DOI: 10.1016/j.ceb.2004.02.007. View