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In Vivo Brain GPCR Signaling Elucidated by Phosphoproteomics Jeffrey J

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In Vivo Brain GPCR Signaling Elucidated by Phosphoproteomics Jeffrey J RESEARCH ◥ of synaptic proteins in the striatum after 5 min RESEARCH ARTICLE SUMMARY of agonist stimulation was partially blocked by protein phosphatase 2A (PP2A) inhibitors, underscoring the involvement of PP2A in KOR- SIGNAL TRANSDUCTION mediated synaptic functions. Pathway analysis revealed enrichment of mTOR (mechanistic target of rapamycin) signaling by agonists In vivo brain GPCR signaling associated with aversion. Strikingly, mTOR inhibition during KOR ◥ elucidated by phosphoproteomics ON OUR WEBSITE activation abolished aver- Read the full article sion while preserving ther- Jeffrey J. Liu, Kirti Sharma, Luca Zangrandi, Chongguang Chen, at http://dx.doi. apeutic antinociceptive Sean J. Humphrey, Yi-Ting Chiu, Mariana Spetea, Lee-Yuan Liu-Chen, org/10.1126/ and anticonvulsant effects. Christoph Schwarzer*, Matthias Mann* science.aao4927 Parallel characterization .................................................. of phosphoproteomic changes related to KOR-mediated mTOR acti- INTRODUCTION: The G protein–coupled re- applied this technology to achieve a global over- vation in a cell line model provided additional ceptor (GPCR) superfamily is a major drug view of the architecture of brain phosphopro- mechanistic insights at the level of the signal- target for neurological diseases. Functionally teome in five mouse brain regions, in which we ing cascade. selective agonists activate GPCRs, such as the examined signaling induced by structurally and Downloaded from kappa opioid receptor (KOR), in a pathway- behaviorally diverse agonists. CONCLUSION: We dissected the signaling specific manner that may lead to drugs with pathways associated with desired and un- fewer side effects. For example, KOR agonists RESULTS: Through the quantification of 50,000 desired outcomes of KOR activation in vivo that trigger beneficial antinociceptive, anti- different phosphosites, this approach yielded and applied this knowledge to suppress the pruritic, and anticonvulsant effects while causing abrainregion–specific systems view of the latter. Our work demonstrates the utility of minimal or no undesirable dysphoric, aversive, phosphoproteome, providing a context to un- combining phosphoproteomics with phar- http://science.sciencemag.org/ or psychotomimetic effects would be invaluable derstand KOR signaling in vivo. We observed macological tools and behavioral assess- in light of the current opioid epidemic. How- strong regional specificity of KOR signaling ments as a general approach for studying ever, functional selectivity observed in vitro attributable to differences in protein-protein GPCR signaling in vivo. Together with ap- frequently has little predictive value for behav- interaction networks, neuronal contacts, and propriate in vitro cellular systems, individu- ioral outcomes. the different tissues in neuronal circuitries. al pathways can be characterized in depth, Agonists with distinct signaling profiles elicited providing a rational basis for GPCR drug RATIONALE: Obtaining a systems view of differential dynamic phosphorylation of synap- discovery.▪ GPCR signaling in the brain may overcome tic proteins, thereby linking GPCR signaling to the gap between in vitro pharmacology and the modulation of brain functions. The most in vivo testing. Recent breakthroughs in mass prominent changes occurred on synaptic pro- spectrometry–based proteomics have enabled teins associated with dopaminergic, glutama- The list of author affiliations is available in the full article online. on November 7, 2018 us to quantify tens of thousands of phosphoryl- tergic, and g-aminobutyric acid–mediated *Corresponding author. Email: [email protected] (M.M.); [email protected] (C.S.) ation events simultaneously in a high-throughput (GABAergic) signaling and synaptic vesicle Cite this article as J. J. Liu et al., Science 360, eaao4927 fashion. Using the KOR as a GPCR model, we release. The large-scale dephosphorylation (2018).DOI:10.1126/science.aao4927 in vivo High-throughput GPCR Signaling HTP Phosphoproteomics phosphoproteomics to characterize in vivo brain GPCR p signaling. Subsequent bioinformatic analysis p enables prediction LC-MS/MS and modulation Analgesia Unwanted of downstream Single run, EasyPhos workflow signaling pathways, which are correlated Bioinformatic Analysis Behavioral Outcomes with unwanted Glutamatergic Analgesia Unwanted effect effects but not the Dopaminergic synapse synapse NMDAR mGlu5 Writhing test Conditioned place aversion therapeutic outcome. VGKC 50 *** 400 ## Cav1.2 VSSC EAAT2 40 Grin2b 200 Kcnb1 Grin2a S1691 S1742 Scn1 Kcna2 S21 S24 S1040 S1284 30 S467 S449 S736 S890 S1198 S1459 S505 0 Kcnd2 Kcnh7 20 -200 S17 Gng12 S548 S174 PSD-95 S415 writhe count 10 -400 S1334 S7 S10 Gi-protein S74 0 -600 **** Shank2 S362 Sapap1 difference test pre-post S648 Src TEM TEM S1338 saline saline saline saline saline Plcb1 vehicle vehicle Camk2b S319 U-50,488H U-50,488H T338 T358 CamkII S1199 DAG S687 TEM + U-50,488H TEM + U-50,488H Liu et al., Science 360, 1314 (2018) 22 June 2018 1of1 RESEARCH ◥ able KOR expression (cerebellum) (Fig. 1C). In RESEARCH ARTICLE total, including biological replicates (three per experimental condition) and follow-up experi- ments, we measured more than 300 single-shot SIGNAL TRANSDUCTION label-free phosphoproteomic samples using the high-throughput EasyPhos platform (30)(Fig.1D). Together, this yielded more than 60,000 different In vivo brain GPCR signaling identified phosphosites mapping to ~6700 brain proteins. This is the most comprehensive cover- age of any organ phosphoproteome reported to elucidated by phosphoproteomics date, underscoring the diversity and importance of phosphorylation-based regulation in the brain Jeffrey J. Liu1, Kirti Sharma1, Luca Zangrandi2, Chongguang Chen3, 1 3 4 3 (Fig. 1E). Sean J. Humphrey , Yi-Ting Chiu , Mariana Spetea , Lee-Yuan Liu-Chen , To obtain a general overview of the archi- 2 1,5 Christoph Schwarzer *, Matthias Mann * tecture of the brain phosphoproteome, we per- formed principal components analysis (PCA), – A systems view of G protein coupled receptor (GPCR) signaling in its native environment is which revealed that the phosphoproteomes of central to the development of GPCR therapeutics with fewer side effects. Using the kappa each brain region cluster tightly and are distinct opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics from other regions. This indicates that each re- to investigate signaling induced by structurally diverse agonists in five mouse brain regions. gion possesses a unique phosphoproteome signa- Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo ture. Furthermore, brain regions that share a Downloaded from signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment similar developmental origin exhibit similarities of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing in their phosphoproteome (Fig. 2A, component 2). aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition The region-specific nature of the phosphoproteome during KOR activation abolished aversion while preserving beneficial antinociceptive and is partly driven by the underlying differences in anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a the proteomes, because proteins highly expressed general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation in one brain region (e.g., striatum) often also yield http://science.sciencemag.org/ of behavioral outcomes. prominent phosphopeptides in the same re- gion (Fig. 2, B and C). In particular, expression he G protein–coupled receptor (GPCR) su- heterogeneity at protein, cellular, and neural path- levels of kinases (32)(the“kinome”)correlated perfamily encompasses drug targets in nu- way levels (20). Thus, it is imperative to develop significantly with the abundance of phospho- merous therapeutic areas, including cancer an unbiased approach that directly investigates peptides containing the corresponding linear T (1)andcardiac(2) and neurological (3)dis- GPCR (and specifically KOR) signaling in vivo, motifs (Fig. 2D). Thus, proteome and kinase ex- eases. Stimulation of a single GPCR, such as therebyelucidatingthelargelyelusivedownstream pression in different regions partially shapes the kappa opioid receptor (KOR), often activates signaling consequences of GPCR activation in the brain phosphoproteome. The complexity parallel pathways, each leading to distinctive phar- the brain. within the brain phosphoproteome is in con- macological outcomes. For example, KOR activa- Developments in mass spectrometry (MS)–based trast to the homogeneity of in vitro cell lines. We 4 tion triggers beneficial analgesic ( ), antipruritic proteomics in general, and phosphoproteomics observed that 85% of synaptic proteins were on November 7, 2018 (5, 6), and anticonvulsant/antiepileptic (7)signaling in particular, now enable the near-comprehensive phosphorylated, which is of particular interest (8) as well as undesirable dysphoria or aversion characterization of proteomes and tens of thou- given previous reports of the importance of phos- and psychotomimetic effects (8, 9)(Fig.1A).Re- sands of phosphorylation events (21–24). Appli- phorylation in the regulation of synaptic plas- cently, some KOR agonists were described that cation of MS to study GPCR signaling has been ticity (33). do not induce conditioned place aversion (CPA) limited to assaying
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