ABSTRACT
Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.
Subject(s)
Biotinylation , Proteome , Proteomics , Proteomics/methods , Reproducibility of Results , Humans , Proteome/metabolism , Mass Spectrometry/methods , HEK293 CellsABSTRACT
Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.
ABSTRACT
Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.
Subject(s)
Cilia , Receptors, Opioid, mu , Receptors, Opioid, mu/metabolism , Cilia/metabolism , Animals , Mice , Humans , HEK293 Cells , Protein TransportABSTRACT
The µ-opioid receptor (µOR) represents an important target of therapeutic and abused drugs. So far, most understanding of µOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet µOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated µOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the µOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new µOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.
ABSTRACT
Many G protein-coupled receptors (GPCRs) initiate a second phase of stimulatory heterotrimeric G protein (Gs)-coupled cAMP signaling after endocytosis. The prevailing current view is that the endosomal signal is inherently ß-arrestin-dependent because ß-arrestin is necessary for receptor internalization and, for some GPCRs, to prolong the endosomal signal. Here we revise this view by showing that the vasoactive intestinal peptide receptor 1 (VIPR1), a secretin-family polypeptide hormone receptor, does not require ß-arrestin to internalize or to generate an endosomal signal. ß-Arrestin instead resolves the plasma membrane and endosomal signaling phases into sequential cAMP peaks by desensitizing the plasma membrane phase without affecting the endosomal phase. This appears to occur through the formation of functionally distinct VIPR1-ß-arrestin complexes at each location that differ in their phosphorylation dependence. We conclude that endosomal GPCR signaling can occur in the absence of ß-arrestin and that ß-arrestin sculpts the spatiotemporal profile of cellular GPCR-G protein signaling through location-specific remodeling of GPCR-ß-arrestin complexes.
Subject(s)
Peptide Hormones , Signal Transduction , beta-Arrestins , beta-Arrestin 1 , Cell MembraneABSTRACT
The cAMP cascade is widely recognized to transduce its physiological effects locally through spatially limited cAMP gradients. However, little is known about how the adenylyl cyclase enzymes, which initiate cAMP gradients, are localized. Here we answer this question in physiologically relevant striatal neurons and delineate how AC localization impacts downstream signaling functions. We show that the major striatal AC isoforms are differentially sorted between ciliary and extraciliary domains of the plasma membrane, and that AC9 is uniquely targeted to endosomes. We identify key sorting determinants in the N-terminal cytoplasmic domain responsible for isoform-specific localization. We also show that AC9-containing endosomes accumulate activated dopamine receptors and form an elaborately intertwined network with juxtanuclear PKA stores bound to Golgi membranes. Finally, we show that endosomal localization is critical for AC9 to selectively elevate PKA activity in the nucleus relative to the cytoplasm. These results reveal a precise spatial landscape of the cAMP cascade in neurons and a key role of AC localization in directing downstream signal transduction to the nucleus.
ABSTRACT
ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.
Subject(s)
GTP-Binding Proteins , Gene Expression Regulation , Receptors, Adrenergic, beta-2 , beta-Arrestins , Humans , beta-Arrestin 1/genetics , beta-Arrestin 1/metabolism , beta-Arrestin 2/genetics , beta-Arrestin 2/metabolism , beta-Arrestins/genetics , beta-Arrestins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation/genetics , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , HEK293 Cells , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , Protein Structure, Tertiary , Protein Isoforms , Enzyme Activation/geneticsABSTRACT
Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.
ABSTRACT
Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental area (VTA). A subset of these neurons expresses the neurotensin receptor NTSR1 and its putative ligand neurotensin (Nts). NTSR1, a G protein-coupled receptor (GPCR), which classically activates Gαq/calcium signaling, is a potential route for modulating DA activity. Drug development efforts have been hampered by the receptor's complex pharmacology and a lack of understanding about its endogenous location and signaling responses. Therefore, we have generated NTSR1-Venus knock-in (KI) mice to study NTSR1 receptors in their physiological context. In primary hippocampal neurons, we show that these animals express functional receptors that respond to agonists by increasing intracellular calcium release and trafficking to endosomes. Moreover, systemic agonist administration attenuates locomotion in KIs as it does in control animals. Mapping receptor protein expression at regional and cellular levels, located NTSR1-Venus on the soma and dendrites of dopaminergic SN/VTA neurons. Direct monitoring of receptor endocytosis, as a proxy for activation, enabled profiling of NTSR1 agonists in neurons, as well as acute SN/VTA containing brain slices. Taken together, NTSR1-Venus animals express traceable receptors that will improve understanding of NTSR1 and DA activities and more broadly how GPCRs act in vivo.
ABSTRACT
Opioid tolerance is well-described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here, we delineate a cellular mechanism underlying this longer-term form of opioid tolerance in cultured rat medium spiny neurons. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the µ-opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptor's cytoplasmic tail, and that partial or biased agonist drugs with reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the δ-opioid receptor (DOR) conforms to the same general paradigm except that presynaptic endocytosis of DOR, in contrast to MOR, does not require phosphorylation of the receptor's cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.
Subject(s)
Analgesics, Opioid , Receptors, Opioid, delta , Rats , Animals , Analgesics, Opioid/pharmacology , Drug Tolerance , Signal Transduction , Endocytosis/physiologyABSTRACT
Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.
Subject(s)
Arrestins , Phosphatidylinositols , beta-Arrestins/metabolism , Phosphatidylinositols/metabolism , Arrestins/metabolism , beta-Arrestin 1/metabolism , Receptors, G-Protein-Coupled/metabolismABSTRACT
ß-Arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G-protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of ß-arrestins is ligand dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical 'mode' for GPCR-mediated triggering of the endocytic activity is presently known - displacement of the ß-arrestin C-terminus (CT) to expose clathrin-coated pit-binding determinants that are masked in the inactive state. Here, we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the ß-arrestin CT and, instead, requires the cytosolic base of the ß-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind ß-arrestin transiently ('class A') primarily triggering the CLB-dependent mode and GPCRs that bind more stably ('class B') triggering both the CT and CLB-dependent modes in combination. Moreover, we show that different modes have opposing effects on the net signaling output of receptors - with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how ß-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.
Subject(s)
Arrestins , Signal Transduction , beta-Arrestins/metabolism , Arrestins/metabolism , Ligands , Signal Transduction/physiology , Receptors, G-Protein-Coupled/metabolism , Arrestin/metabolismABSTRACT
The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gßγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.
Subject(s)
Peptides , Receptors, G-Protein-Coupled , GTP Phosphohydrolases , Guanine Nucleotides , Nucleotides , Peptides/chemistry , Peptides, Cyclic/pharmacologyABSTRACT
A number of G protein-coupled receptors (GPCRs) are now thought to use endocytosis to promote cellular cAMP signaling that drives downstream transcription of cAMP-dependent genes. We tested if this is true for the glucagon receptor (GCGR), which mediates physiological regulation of hepatic glucose metabolism via cAMP signaling. We show that epitope-tagged GCGRs undergo clathrin- and dynamin-dependent endocytosis in HEK293 and Huh-7-Lunet cells after activation by glucagon within 5 min and transit via EEA1-marked endosomes shown previously to be sites of GPCR/Gs-stimulated production of cAMP. We further show that endocytosis potentiates cytoplasmic cAMP elevation produced by GCGR activation and promotes expression of phosphoenolpyruvate carboxykinase 1 (PCK1), the enzyme catalyzing the rate-limiting step in gluconeogenesis. We verify endocytosis-dependent induction of PCK1 expression by endogenous GCGRs in primary hepatocytes and show similar control of two other gluconeogenic genes (PGC1α and G6PC). Together, these results implicate the endosomal signaling paradigm in metabolic regulation by glucagon.
Subject(s)
Gluconeogenesis , Receptors, Glucagon , Endocytosis , Gene Expression Regulation , Glucagon/genetics , Glucagon/metabolism , Glucagon/pharmacology , Gluconeogenesis/genetics , HEK293 Cells , Hepatocytes/metabolism , Humans , Liver/metabolism , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Receptors, Glucagon/genetics , Receptors, Glucagon/metabolism , Transcription, GeneticABSTRACT
Adaptive neuronal circuit function requires a continual adjustment of synaptic network parameters known as "neuromodulation." This process is now understood to be based primarily on the binding of myriad secreted "modulatory" ligands such as dopamine, serotonin and the neuropeptides to G protein-coupled receptors (GPCRs) that, in turn, regulate the function of the ion channels that establish synaptic weights and membrane excitability. Many of the basic molecular mechanisms of neuromodulation are now known, but the organization of neuromodulation at a network level is still an enigma. New single-cell RNA sequencing data and transcriptomic neurotaxonomies now offer bright new lights to shine on this critical "dark matter" of neuroscience. Here we leverage these advances to explore the cell-type-specific expression of genes encoding GPCRs, modulatory ligands, ion channels and intervening signal transduction molecules in mouse hippocampus area CA1, with the goal of revealing broad outlines of this well-studied brain structure's neuromodulatory network architecture.
Subject(s)
Hippocampus , Neurons , Animals , Hippocampus/physiology , Ion Channels/metabolism , Ligands , Mice , Neurons/physiology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolismABSTRACT
Nuclear entry of cAMP-dependent protein kinase catalytic subunits is typically inferred from changes in net protein amount or kinase activity in the nucleus. Previous methods to directly assess nuclear entry require kinase subunit overexpression and/or supraphysiological cAMP elevation. We describe a method to detect nuclear entry of catalytic subunits expressed at an endogenous level in living cells, stimulated by cAMP in a physiological range, and in real time.
Subject(s)
Cell Nucleus , Cyclic AMP-Dependent Protein Kinases , Biological Assay , Catalytic Domain , Research DesignABSTRACT
Endosomal signaling downstream of G-protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. However, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass-spectrometry-based analysis of the phosphoproteome. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP elevation. We next determined that the same amount of cAMP produced from the endosomal membrane led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of 218 identified targets and irrespective of their annotated subcellular localizations (endosome, cell surface, nucleus, cytosol). Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A by cAMP-responsive kinases as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness to cAMP by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.
Subject(s)
Cyclic AMP/metabolism , Endosomes/metabolism , Phosphoproteins/metabolism , Proteome/metabolism , Animals , HEK293 Cells , Humans , Phosphoproteins/chemistry , Phosphorylation , Protein Domains , Protein Phosphatase 2/metabolism , Proteome/chemistryABSTRACT
G-protein-coupled receptor-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its overall amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale, whereas endosomes typically localize micrometers from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunits to direct downstream signaling.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Cytoplasm/metabolism , Endosomes/metabolism , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/genetics , Animals , Catalytic Domain , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Clathrin Heavy Chains/antagonists & inhibitors , Clathrin Heavy Chains/genetics , Clathrin Heavy Chains/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cytoplasm/ultrastructure , Dynamin I/genetics , Dynamin I/metabolism , Endosomes/ultrastructure , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Holoenzymes/genetics , Holoenzymes/metabolism , Humans , Luciferases/genetics , Luciferases/metabolism , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Receptors, Adrenergic, beta-2/geneticsABSTRACT
Intricate relationships between endocytosis and cellular signaling, first recognized nearly 40 years ago through the study of tyrosine kinase growth factor receptors, are now known to exist for multiple receptor classes and to affect myriad physiological and developmental processes. This review summarizes our present understanding of how endocytosis orchestrates cellular signaling networks, with an emphasis on mechanistic underpinnings and focusing on two receptor classes-tyrosine kinase and G protein-coupled receptors-that have been investigated in particular detail. Together, these examples provide a useful survey of the current consensus, uncertainties, and controversies in this rapidly advancing area of cell biology.
Subject(s)
Endocytosis/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Cell Membrane/metabolism , Endosomes/metabolism , Humans , Lysosomes/metabolism , Protein Transport , Signal TransductionABSTRACT
Advances in proteomic methodologies based on quantitative mass spectrometry are now transforming pharmacology and experimental biology more broadly. The present review will discuss several examples based on work in the author's laboratory, which focuses on delineating relationships between G protein-coupled receptor signaling and trafficking in the endocytic network. The examples highlighted correspond to those discussed in a talk presented at the 2019 EB/ASPET meeting, which was organized by Professor Joe Beavo to commemorate his receipt of the Julius Axelrod Award. SIGNIFICANCE STATEMENT: GPCRs are allosteric machines that signal by interacting with other cellular proteins, and this, in turn, is determined by a complex interplay between the biochemical, subcellular localization, and membrane trafficking properties of receptors relative to transducer and regulatory proteins. The present minireview highlights recent advances and challenges in elucidating this dynamic cell biology and toward delineating the cellular basis of drug action at the level of defined GPCR interaction networks using proteomic approaches enabled by quantitative mass spectrometry.