Article pubs.acs.org/biochemistry
Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gβ and Gγ Subunits in C57Bl6/J Brain Synaptosomes Yun Young Yim,† W. Hayes McDonald,‡ Karren Hyde,† Osvaldo Cruz-Rodríguez,§,∥,⊥ John J. G. Tesmer,§,∥ and Heidi E. Hamm*,† †
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6600, United States Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232-6600, United States § Life Sciences Institute, ∥Departments of Pharmacology and Biological Chemistry, and ⊥Ph.D. Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109, United States ‡
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, Gβ1 was mostly localized at the membrane while Gβ2 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.
not only the regulatory effects of Gβγ dimer specificity in physiology but also disease pathophysiology such as depression, ADHD, and Parkinson’s disease.17 In mammals, there are five different Gβ genes and 12 different Gγ genes encoding each subunit.18−20 Gβ1−4 share up to 90% amino acid sequence identity, whereas Gβ5 is only 50% identical.21,22 In contrast, Gγ subunits are very divergent, sharing only 30−70% sequence identity.21,22 Made up of two αhelices, Gγ subunits can be post-translationally modified at the processed C-terminal cysteine that is carboxymethylated and modified with a farnesyl or geranylgeranyl moiety via a thioether bond. These modifications aid Gβγ dimers in membrane localization.23,24 Together, Gβ and Gγ subunits form Gβγ dimers and, once assembled, act as signaling units for GPCRs. Although we do not fully understand the selectivity of Gβγ dimers for various GPCRs and effectors, some studies have hypothesized that Gβ subunits determine the Gβγ−effector specificity, while Gγ subunits confer Gβγ−receptor specificity.3,22,25−31 To date, numerous genetic deletion studies and knockout animal studies have suggested specific roles for
G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane proteins, encoded by approximately 800 genes in the human genome, and are some of the most successful drug targets.1,2 Upon activation, GPCRs transduce extracellular signals into various intracellular responses by the activation and dissociation of heterotrimeric G proteins. Heterotrimeric G proteins, made up of Gα, Gβ, and Gγ subunits, dissociate after activation of GPCRs into a GTPbound Gα (GTP-Gα) and a Gβγ dimer; each signals to a number of effectors. Gβγ dimers interact with adenylyl cyclases and phospholipase Cβ, in addition to PI3-kinase and components of the mitogen-activated protein kinase cascade.3−8 In the central nervous system (CNS), Gβγ dimers also interact with voltage-dependent calcium (VDCC) and inward-rectifying potassium (GIRK) channels and soluble NSF attachment proteins (SNARE) to regulate neurotransmitter release at the synapse.9−16 Although many of these Gβγ− effector interactions and downstream signaling cascades are well-understood, it is still unclear which combination of Gβγ dimers is present in vivo and what factors control the specificity of Gβγ dimers to their effectors. Given the diversity of GPCRs, G proteins, and Gβγ effectors, and the importance of GPCRs as drug targets, examining the expression and subcellular localization of Gβ and Gγ subunits will aid in our understanding of © XXXX American Chemical Society
Received: May 5, 2017 Revised: August 29, 2017 Published: September 7, 2017 A
DOI: 10.1021/acs.biochem.7b00433 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry different Gβ and Gγ subunits in intact cells and mice.30,32 Various Gβ and Gγ subunits are implicated in neurodevelopmental disability, hypotonia, and seizures.33−35 These unique physiological phenotypes of each Gβ and Gγ subunit suggest a great deal of specificity in Gβγ dimerization and signaling.36−38 In situ hybridization studies of Gβ and Gγ subunits in the CNS indicate how the distribution of Gβ and Gγ subunits may affect Gβγ dimerization in specific brain regions and cell types.7,21,22 Some Gβ and Gγ subunits are ubiquitously expressed, whereas others are localized in specific brain regions and cell types.21,39−43 Because most cell types express multiple Gβ and Gγ subunits, specific expression levels and localization may influence intracellular signaling cascades through the formation of specific Gβγ dimers. Although there are 60 different theoretical combinations of Gβγ dimers,44,45 numerous in vitro assays and yeast-two hybrid analyses have indicated that not all theoretical Gβγ dimers exist, are equally expressed, or interact with Gα subunits, receptors, effectors, or downstream signaling factors.19,32,35,46−52 Each Gβ and Gγ subunit shows widely varying affinities for one another.19,22,53 While Gβ1 and Gβ4 dimerize with all Gγ subunits, Gβ2 and Gβ3 are unable to dimerize with Gγ1 and Gγ11.54 Gβ2γ1 shows a stronger association than Gβ2γ4.22,34,55 Different affinities between Gβ and Gγ subunits, in combination with the expression and localization of individual Gβ and Gγ subunits, may determine which Gβγ dimers are active in a given cell.21 Interestingly, various Gβγ dimers have been reported to have different affinities for their effectors. Gβ1γ2 has a 40-fold higher affinity for SNARE and a 20-fold higher level of inhibition of exocytosis than Gβ1γ1 does.56 We speculate that the difference in the Gγ subunit, per se, or in the post-translational modification of Gγ1 and Gγ2 may cause the change in affinity. Such expression and affinity diversity of Gβ and Gγ subunits and the affinity of Gβγ−effector interactions may also suggest that specific dimers could permit specialized roles in signal transduction pathways through association with particular GPCRs. For example, Gβ2γ and Gβ4γ dimers may specifically interact with adrenergic and opioid GPCRs while Gβ1γ and Gβ3γ dimers, particularly Gβ1γ3 and Gβ3γ4, may preferentially couple with somatostatin and muscarinic M4 GPCRs.57−59 Thus, a greater understanding of the expression and subcellular localization of Gβ and Gγ subunits in the CNS will be particularly important in determining the physiologically relevant Gβγ dimers, the roles of each unique Gβγ dimer in regulating signaling cascades, and their impact in neurological diseases and GPCR-targeted drug mechanisms. Despite many attempts to quantify the Gβ and Gγ protein level,39,42,43,60−62 it has been difficult to develop reliable subunit-specific detection methods because of the high degree of sequence homology between subunits and the lack of subunit-specific antibodies.63 To overcome this issue, we previously developed a targeted multiple-reaction monitoring (MRM)64,65 mass spectrometric approach to identify neuronal Gβ and Gγ subunits. We found a regional and subcellular expression pattern of four Gβ (Gβ1, Gβ2, Gβ4, and Gβ5) and six Gγ (Gγ2−Gγ4, Gγ7, Gγ12, and Gγ13) subunits in mouse crude synaptosomes of cortex, cerebellum, hippocampus, and striatum.62 However, we were unable to quantify and compare the expression level of each Gβ and Gγ subunit. Here, we use the quantitative MRM method of Gβ and Gγ subunits, in combination with the isotopic labeling of standards and Skyline analysis,62,66 to generate a comprehensive, quantitative brain map of Gβ and Gγ subunits. We measured and compared the
protein level and subcellular localization of neuronal Gβ and Gγ subunits and predicted the in vivo expression level of Gβγ dimers, further supporting the Gβγ specificity to particular GPCRs and effector proteins to maintain normal brain function.
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MATERIALS AND METHODS
Synaptosome Preparation. All animal handling and procedures were conducted in accordance with the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Vanderbilt Institutional Animal Care and Use Committee. Crude synaptosomes were made from adult, male C57Bl6/J mice as described previously.62,67,68 Briefly, whole brains were homogenized in 20 mL of a 0.32 M sucrose solution [0.32 M sucrose, 4.2 mM HEPES (pH 7.4), 0.1 mM CaCl2, 1 mM MgCl2, 1.54 μM aprotinin, 10.7 μM leupeptin, 0.95 μM pepstatin, and 200 μM PMSF]. Homogenates were centrifuged at 1000g and 4 °C for 10 min, and supernatants containing synaptosomes (S1) were transferred to clean conical tubes. Pellets were resuspended in 20 mL of a 0.32 M sucrose solution and centrifuged again. Pellets were discarded. Supernatants (S1) were combined and centrifuged at 10000g and 4 °C for 20 min to produce the crude synaptosome pellet. Crude synaptosomes were stored at −80 °C. Synaptosome Lysate. Crude synaptosomes were gently resuspended in 4 mL of RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 1.54 μM aprotinin, 10.7 μM leupeptin, 0.948 μM pepstatin, and 200 μM PMSF] using a 25 gauge needle to lyse membranes. Lysate concentrations were determined with a BCA assay kit (Pierce) and diluted to 1 mg/mL using RIPA buffer. The diluted homogenate was placed on a rotator for 1 h and maintained at 4 °C. Homogenates were transferred to a 2 mL Eppendorf tube and centrifuged at 14000 rpm and 4 °C for 10 min to separate the triton soluble and insoluble fractions. Supernatants, the triton soluble fractions, were collected. Protein concentrations were determined with a BCA assay kit (Pierce). Subcellular Fractionation. Subcellular fractions were prepared as previously described62,69 (Figure 4). Briefly, crude synaptosomes were gently resuspended in 4 mL of hypotonic lysis buffer [20 mM Tris (pH 6.0), 0.1 mM CaCl2, 1 mM MgCl2, 1% Triton X-100, 1.54 μM aprotinin, 10.7 μM leupeptin, 0.95 μM pepstatin, and 200 μM PMSF] and incubated on ice for 20 min to lyse membranes. Lysates were subjected to ultracentrifugation at 100000g and 4 °C for 2 h using a SW-55 Ti rotor (Beckman Coulter) to separate supernatants consisting of the synaptosomal cytosolic fractions from membrane fractions. The synaptosomal cytosolic fraction may contain crude vesicles.70 Supernatants were transferred to clean conical tubes. Pellets contacting membrane fractions were resuspended in 2 mL of Tris pH 8.0 buffer [20 mM Tris (pH 8.0), 1% Triton X-100, 1.54 μM aprotinin, 10.7 μM leupeptin, 0.95 μM pepstatin, and 200 μM PMSF] and incubated on ice for 20 min. Lysates were centrifuged at 10000g and 4 °C for 30 min, and supernatants containing enriched presynaptic fractions were collected. Finally, pellets were resuspended in 400 μL of a 1× PBS/0.5% SDS buffer and centrifuged at 10000g and 4 °C for 30 min. Supernatants containing enriched postsynaptic fractions were collected. Pellets were discarded. Protein concentrations of each fraction were determined with a BCA assay kit (Pierce). B
DOI: 10.1021/acs.biochem.7b00433 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry Table 1. List of Heavy Labeled Proteotypic Peptides name Gβ1 Gβ2 Gβ4 Gβ5 Gγ2 Gγ3 Gγ4 Gγ7 Gγ12 Gγ13
sequence position
peptide sequence
precursor m/z
charge
198−209 284−301 198−209 257−280 198−209 305−314 129−139 318−327 21−27 47−62 3−17 25−31 3−17 51−66 19−25 45−60 5−15 23−29 18−23 37−44
(R)LFVSGACDASAK(L) (R)LLLAGYDDFNCNVWDALK (A) (R)TFVSGACDASIK(L) (R)ADQELLMYSHDNIICGITSVAFSR(S) (R)TFVSGACDASSK(L) (R)SGVLAGHDNR(V) (K)VIVWDSFTTNK(E) (R)VSILFGHENR(V) (K)MEANIDR(I) (K)EDPLLTPVPASENPFR(E) (K)GETPVNSTMSIGQAR(K) (K)IEASLCR(I) (K)EGMSNNSTTSISQAR(K) (R)EDPLIIPVPASENPFR(E) (R)IEAGIER(I) (R)NDPLLVGVPASENPFK(D) (K)TASTNSIAQAR(R) (R)LEASIER(I) (K)YQLAFK(R) (K)WIEDGIPK(D)
617.3048 1068.0215 632.3101 917.4432/922.7749 619.2841 518.2639/345.8450 659.3501 591.3187 429.7043/437.7018 896.4612 779.3819/787.3793 429.7225 796.8641/804.8615 902.4794 399.2232 852.9560 565.2954 414.2285 389.2229 483.2627
2 2 2 3 2 2/3 2 2 2 2 2 2 2 2 2 2 2 2 2 2
product ion m/z 487.23, 640.35, 508.25, 419.23, 495.23, 340.16, 705.37, 498.27, 527.28, 774.43, 441.24, 960.48, 471.25, 927.46, 484.28, 738.42, 655.38, 514.29, 373.23, 252.18,
787.35, 874.38, 973.45 898.40, 954.94, 1127.54 829.40, 916.43, 1015.50 490.26, 676.37, 777.41, 949.52 675.29, 803.34, 890.38, 989.44 396.70, 446.24, 474.75, 608.28, 679.31, 792.40 820.39, 1006.47, 1105.54 622.29, 769.36, 882.45 598.32, 727.36 927.46, 1123.58, 1224.62 554.33, 635.83, 643.82, 686.35,694.35 1074.52, 1090.5i, 458.24, 545.27, 616.31, 745.35 584.34, 671.37, 873.47, 960.50 1123.58, 1236.66 555.31, 684.36 897.46, 1053.55, 1152.61 769.42, 957.50 585.32, 714.37 486.32, 614.38 422.29, 537.31, 666.35
a Bold arginine and lysine residues were heavy labeled with 13C or 15N. Bold sequence positions indicate proteotypic peptides with the strongest fragmentation ion intensity. These peptides were used for the heavy labeled proteotypic peptide detection analysis (data not shown).
Heavy Labeled Proteotypic Peptide Detection Analysis. To examine the detection of each heavy labeled proteotypic peptide, we selected one heavy labeled peptide with the largest area under the curve from the work of Betke et al.62 (bold in Table 1), for each Gβ and Gγ subunit (Table 1); 0.04, 0.2, 0.4, 4, 20, 40, and 100 fmol of each heavy labeled peptide were pooled and mixed with bovine serum albumin (BSA), analyzed by TSQ Vantage triple-quadrupole mass spectrometry (Thermo Scientific), and quantified using Skyline66 (data not shown). Heavy Labeled Peptide Cocktail. On the basis of the amino acid sequence of tryptically digested proteins and fragment ion signal intensities in the previous study,62 we have selected two proteotypic peptides for four Gβ (Gβ1, Gβ2, Gβ4, and Gβ5) and six Gγ (Gγ2−Gγ4, Gγ7, Gγ12, and Gγ13) subunits detected in crude synaptosomes. Proteotypic peptides listed in Table 1 were synthesized via SPOT synthesis (JPT Peptide Technologies). The arginine or lysine at the C-termini of these peptides was isotopically labeled with heavy 13C or 15N with a trypsin cleavable Qtag to quantify (JPT). Heavy labeled peptides are 8−10 Da heavier without a change in the physiological properties and chemical reactivity than the nonlabeled proteolytic peptides. One nanomole of each peptide was resolubilized in 200 μL of high-performance liquid chromatography (HPLC) grade water and 200 μL of 10% acetonitriles to make a stock concentration of 5 pM/μL. According to the mass spectrometry signal strength of each proteotypic peptide, different amounts of peptides were pooled to create a “heavy labeled peptide cocktail”. The cocktails were mixed with BSA, 1.5 M Tris, and HPLC grade water before being reduced with 2.5 mM TCEP for 30 min and alkylated with 5 mM iodoacetamide for 30 min in the dark. The cocktail was then digested overnight with 0.5 mg of trypsin and then stored at −80 °C. Before each MRM analysis, the cocktail was acidified with 1.5 μL of formic acid and added to samples as internal standards for quantification.
Antibodies. Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, MAB374, 1:20000), mouse anti-SNAP25 (Santa Cruz, sc-376713, 1:500), mouse antisyntaxin-1 (Santa Cruz, sc-12736, 1:2000), mouse anti-Nmethyl-D-aspartate receptor-1 (NMDAR1) (BD Pharmingen, 556308, 1:2000), mouse anti-postsynaptic density-95 (PSD-95) (Neuromab, 75-028, 1:20000), and rabbit anti-Gβ (Santa Cruz, sc-378, 1:15000) were used. HRP-conjugated secondary antibodies were obtained from PerkinElmer and Jackson Immunoresearch and used at the following dilutions: goat anti-rabbit (1:20000), goat anti-mouse (1:10000 for NMDAR1 and syntaxin and 1:20000 for PDS-95, GAPDH, and SNAP25), and mouse anti-rabbit light chain-specific (1:7500, Gβ). Immunoblot Analysis. To examine the fractionation of crude synaptosomes, Western blot analysis was performed on 7 μg of synaptosomal cytosolic, presynaptic, and postsynaptic fractions as described previously.62 Protein Purification of Gβγ Dimers. Gβ1γ1 was purified from the bovine retina as described previously.71 Recombinant His6-tagged Gβ5γ2 was expressed in Sf9 cells and purified using nickel-nitrilotriacetic acid affinity chromatography (Sigma-100 Aldrich, St. Louis, MO). Human Gβ1γ2, containing an Nterminally hexahistidine-tagged β subunit, was expressed in High Five cells using a dual promoter insect cell expression vector described previously.72 Gβ1γ2 was purified from membrane extracts of High Five cells harvested 48 h postinfection using nickel-nitrilotriacetic acid affinity and anion exchange chromatography as described previously.73 Fractions containing Gβ1γ2 were subsequently pooled and buffer exchanged into 20 mM HEPES (pH 8.0), 100 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, and 1 mM DTT using a S200 column. Gβ1γ2 fractions were then concentrated to 5 mg/mL, as determined by Bradford analysis, in a 30 kDa cutoff Amicon Ultra-15 Centrifugal Filter Unit, flash-frozen in liquid nitrogen, and stored at −80 °C until future use. C
DOI: 10.1021/acs.biochem.7b00433 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry Quantitative MRM of Gβ and Gγ Subunits. As shown in Figure 2, samples containing Gβ and Gγ subunits were separated by 12.5% acrylamide sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) and stained with colloidal Coomassie Blue (Invitrogen). Using Gβ1γ1 and Gβ5γ2 as markers, Gβ and Gγ bands were excised and in-gel digested as described previously62 and then resolubilized in the presence of the “heavy labeled peptide cocktail” and run on a TSQ vantage triple-quadrupole mass spectrometer (Thermo Scientific) following the scheduled MRM method as described previously.62 Gβ and Gγ subunits were run separately, and data were analyzed in Skyline. Correct peaks were manually chosen on the basis of retention time, dot plot values, relative distributions of the transition ion, and heavy labeled peptide peaks. Samples were dropped from analysis if no correct peaks could be chosen. Furthermore, all chosen peaks with a signalto-noise (S/N) ratio of
