Research Article

In vivo brain GPCR signaling elucidated by phosphoproteomics

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Science  22 Jun 2018:
Vol. 360, Issue 6395, eaao4927
DOI: 10.1126/science.aao4927

Mechanisms of drug action

Advanced mass spectrometry methods enable monitoring of tens of thousands of phosphorylation sites in proteins. This technology can potentially distinguish cellular signaling pathways that produce beneficial effects from those that produce unwanted side effects. Liu et al. treated mice with various agonists of the kappa opioid receptor (a G protein–coupled receptor) and monitored changes in phosphorylation over time in different brain regions. The phosphorylation patterns revealed distinct patterns of signaling in various brain tissues, some of which were associated with unwanted side effects.

Science, this issue p. eaao4927

Structured Abstract


The G protein–coupled receptor (GPCR) superfamily is a major drug target for neurological diseases. Functionally selective agonists activate GPCRs, such as the kappa opioid receptor (KOR), in a pathway-specific manner that may lead to drugs with fewer side effects. For example, KOR agonists that trigger beneficial antinociceptive, antipruritic, and anticonvulsant effects while causing minimal or no undesirable dysphoric, aversive, or psychotomimetic effects would be invaluable in light of the current opioid epidemic. However, functional selectivity observed in vitro frequently has little predictive value for behavioral outcomes.


Obtaining a systems view of GPCR signaling in the brain may overcome the gap between in vitro pharmacology and in vivo testing. Recent breakthroughs in mass spectrometry–based proteomics have enabled us to quantify tens of thousands of phosphorylation events simultaneously in a high-throughput fashion. Using the KOR as a GPCR model, we applied this technology to achieve a global overview of the architecture of brain phosphoproteome in five mouse brain regions, in which we examined signaling induced by structurally and behaviorally diverse agonists.


Through the quantification of 50,000 different phosphosites, this approach yielded a brain region–specific systems view of the phosphoproteome, providing a context to understand KOR signaling in vivo. We observed strong regional specificity of KOR signaling attributable to differences in protein-protein interaction networks, neuronal contacts, and the different tissues in neuronal circuitries. Agonists with distinct signaling profiles elicited differential dynamic phosphorylation of synaptic proteins, thereby linking GPCR signaling to the modulation of brain functions. The most prominent changes occurred on synaptic proteins associated with dopaminergic, glutamatergic, and γ-aminobutyric acid–mediated (GABAergic) signaling and synaptic vesicle release. The large-scale dephosphorylation of synaptic proteins in the striatum after 5 min of agonist stimulation was partially blocked by protein phosphatase 2A (PP2A) inhibitors, underscoring the involvement of PP2A in KOR-mediated synaptic functions. Pathway analysis revealed enrichment of mTOR (mechanistic target of rapamycin) signaling by agonists associated with aversion. Strikingly, mTOR inhibition during KOR activation abolished aversion while preserving therapeutic antinociceptive and anticonvulsant effects. Parallel characterization of phosphoproteomic changes related to KOR-mediated mTOR activation in a cell line model provided additional mechanistic insights at the level of the signaling cascade.


We dissected the signaling pathways associated with desired and undesired outcomes of KOR activation in vivo and applied this knowledge to suppress the latter. Our work demonstrates the utility of combining phosphoproteomics with pharmacological tools and behavioral assessments as a general approach for studying GPCR signaling in vivo. Together with appropriate in vitro cellular systems, individual pathways can be characterized in depth, providing a rational basis for GPCR drug discovery.

High-throughput phosphoproteomics to characterize in vivo brain GPCR signaling.

Subsequent bioinformatic analysis enables prediction and modulation of downstream signaling pathways, which are correlated with unwanted effects but not the therapeutic outcome.


A systems view of G protein–coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.

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