Exploiting Cell-Based Assays to Accelerate Drug Development for G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects.

Protocol for identifying properties of ERBB receptor antagonists using the barcoded ERBBprofiler assay.

The ERBBprofiler assay measures compound effects on ERBB family receptors and key downstream signaling pathways that are implicated in cancer or other complex diseases. Here, we present a protocol for identifying properties of ERBB receptor antagonists using the barcoded ERBBprofiler assay. We describe steps for in-solution transfection, cell treatment, combined processing of samples, amplification and indexing of PCRs, sequencing, and data analysis. This approach allows for the simultaneous assessment of drug effects and cell-type-dependent effects. For complete details on the use and execution of this protocol, please refer to Popovic et al.1.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists.

ERBB receptor tyrosine kinases are involved in development and diseases like cancer, cardiovascular, neurodevelopmental, and mental disorders. Although existing drugs target ERBB receptors, the next generation of drugs requires enhanced selectivity and understanding of physiological pathway responses to improve efficiency and reduce side effects. To address this, we developed a multilevel barcoded reporter profiling assay, termed 'ERBBprofiler', in living cells to monitor the activity of all ERBB targets and key physiological pathways simultaneously. This assay helps differentiate on-target therapeutic effects from off-target and off-pathway side effects of ERBB antagonists. To challenge the assay, eight established ERBB antagonists were profiled. Known effects were confirmed, and previously uncharacterized properties were discovered, such as pyrotinib's preference for ERBB4 over EGFR. Additionally, two lead compounds selectively targeting ERBB4 were profiled, showing promise for clinical trials. Taken together, this multiparametric profiling approach can guide early-stage drug development and lead to improved future therapeutic interventions.

Comprehensive split TEV based protein-protein interaction screening reveals TAOK2 as a key modulator of Hippo signalling to limit growth.

The conserved Hippo signalling pathway plays a crucial role in tumour formation by limiting tissue growth and proliferation. At the core of this pathway are tumour suppressor kinases STK3/4 and LATS1/2, which limit the activity of the oncogene YAP1, the primary downstream effector. Here, we employed a split TEV-based protein-protein interaction screen to assess the physical interactions among 28 key Hippo pathway components and potential upstream modulators. This screen led us to the discovery of TAOK2 as pivotal modulator of Hippo signalling, as it binds to the pathway's core kinases, STK3/4 and LATS1/2, and leads to their phosphorylation. Specifically, our findings revealed that TAOK2 binds to and phosphorylates LATS1, resulting in the reduction of YAP1 phosphorylation and subsequent transcription of oncogenes. Consequently, this decrease led to a decrease in cell proliferation and migration. Interestingly, a correlation was observed between reduced TAOK2 expression and decreased patient survival time in certain types of human cancers, including lung and kidney cancer as well as glioma. Moreover, in cellular models corresponding to these cancer types the downregulation of TAOK2 by CRISPR inhibition led to reduced phosphorylation of LATS1 and increased proliferation rates, supporting TAOK2's role as tumour suppressor gene. By contrast, overexpression of TAOK2 in these cellular models lead to increased phospho-LATS1 but reduced cell proliferation. As TAOK2 is a druggable kinase, targeting TAOK2 could serve as an attractive pharmacological approach to modulate cell growth and potentially offer strategies for combating cancer.

Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

Amyloid Pathology in the Central Auditory Pathway of 5XFAD Mice Appears First in Auditory Cortex.

BACKGROUND: Effective treatment of Alzheimer's disease (AD) will hinge on early detection. This has led to the search for early biomarkers that use non-invasive testing. One possible early biomarker is auditory temporal processing deficits, which reflect central auditory pathway dysfunction and precede cognitive and memory declines in AD. Gap detection is a measure of auditory temporal processing, is impaired in human AD, and is also impaired in the 5XFAD mouse model of AD. Gap detection deficits appear as early as postnatal day 60 in 5XFAD mice, months before cognitive deficits or cell death, supporting gap detection as an early biomarker. However, it remains unclear how gap detection deficits relate to the progression of amyloid pathology in the auditory system. OBJECTIVE: To determine the progression of amyloid pathology throughout the central auditory system and across age in 5XFAD mice. METHODS: We quantified intracellular and extracellular antibody labelling of Aß42 in 6 regions of the central auditory system from p14 to p150. RESULTS: Pathology appeared first in primary auditory cortex (A1) as intracellular accumulation of Aß42 in layer 5 pyramidal neurons by age p21. Extracellular plaques appeared later, by age p90, in A1, medial geniculate body, and inferior colliculus. Auditory brainstem structures showed minimal amyloid pathology. We also observed pathology in the caudal pontine reticular nucleus, a brainstem structure that is outside of the central auditory pathway but which is involved in the acoustic startle reflex. CONCLUSION: These results suggest that Aß42 accumulation, but not plaques, may impair gap detection.

Effects of Locomotion in Auditory Cortex Are Not Mediated by the VIP Network.

Movement has a prominent impact on activity in sensory cortex, but has opposing effects on visual and auditory cortex. Both cortical areas feature a vasoactive intestinal peptide-expressing (VIP) disinhibitory circuit, which in visual cortex contributes to the effect of running. In auditory cortex, however, the role of VIP circuitry in running effects remains poorly understood. Running and optogenetic VIP activation are known to differentially modulate sound-evoked activity in auditory cortex, but it is unknown how these effects vary across cortical layers, and whether laminar differences in the roles of VIP circuitry could contribute to the substantial diversity that has been observed in the effects of both movement and VIP activation. Here we asked whether VIP neurons contribute to the effects of running, across the layers of auditory cortex. We found that both running and optogenetic activation of VIP neurons produced diverse changes in the firing rates of auditory cortical neurons, but with distinct effects on spontaneous and evoked activity and with different patterns across cortical layers. On average, running increased spontaneous firing rates but decreased evoked firing rates, resulting in a reduction of the neuronal encoding of sound. This reduction in sound encoding was observed in all cortical layers, but was most pronounced in layer 2/3. In contrast, VIP activation increased both spontaneous and evoked firing rates, and had no net population-wide effect on sound encoding, but strongly suppressed sound encoding in layer 4 narrow-spiking neurons. These results suggest that VIP activation and running act independently, which we then tested by comparing the arithmetic sum of the two effects measured separately to the actual combined effect of running and VIP activation, which were closely matched. We conclude that the effects of locomotion in auditory cortex are not mediated by the VIP network.

Auditory Cortex Contributes to Discrimination of Pure Tones.

The auditory cortex is topographically organized for sound frequency and contains highly selective frequency-tuned neurons, yet the role of auditory cortex in the perception of sound frequency remains unclear. Lesion studies have shown that auditory cortex is not essential for frequency discrimination of pure tones. However, transient pharmacological inactivation has been reported to impair frequency discrimination. This suggests the possibility that successful tone discrimination after recovery from lesion surgery could arise from long-term reorganization or plasticity of compensatory pathways. Here, we compared the effects of lesions and optogenetic suppression of auditory cortex on frequency discrimination in mice. We found that transient bilateral optogenetic suppression partially but significantly impaired discrimination performance. In contrast, bilateral electrolytic lesions of auditory cortex had no effect on performance of the identical task, even when tested only 4 h after lesion. This suggests that when auditory cortex is destroyed, an alternative pathway is almost immediately adequate for mediating frequency discrimination. Yet this alternative pathway is insufficient for task performance when auditory cortex is intact but has its activity suppressed. These results indicate a fundamental difference between the effects of brain lesions and optogenetic suppression, and suggest the existence of a rapid compensatory process possibly induced by injury.

Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays.

Receptor tyrosine kinases (RTKs) play key roles in various aspects of cell biology, including cell-to-cell communication, proliferation and differentiation, survival, and tissue homeostasis, and have been implicated in various diseases including cancer and neurodevelopmental disorders. Ligand-activated RTKs recruit adapter proteins through a phosphotyrosine (p-Tyr) motif that is present on the RTK and a p-Tyr-binding domain, like the Src homology 2 (SH2) domain found in adapter proteins. Notably, numerous combinations of RTK/adapter combinations exist, making it challenging to compare receptor activities in standardised assays. In cell-based assays, a regulated adapter recruitment can be investigated using genetically encoded protein-protein interaction detection methods, such as the split TEV biosensor assay. Here, we applied the split TEV technique to robustly monitor the dynamic recruitment of both naturally occurring full-length adapters and artificial adapters, which are formed of clustered SH2 domains. The applicability of this approach was tested for RTKs from various subfamilies including the epidermal growth factor (ERBB) family, the insulin receptor (INSR) family, and the hepatocyte growth factor receptor (HGFR) family. Best signal-to-noise ratios of ligand-activated RTK receptor activation was obtained when clustered SH2 domains derived from GRB2 were used as adapters. The sensitivity and robustness of the RTK recruitment assays were validated in dose-dependent inhibition assays using the ERBB family-selective antagonists lapatinib and WZ4002. The RTK split TEV recruitment assays also qualify for high-throughput screening approaches, suggesting that the artificial adapter may be used as universal adapter in cell-based profiling assays within pharmacological intervention studies.

Pathway sensor-based functional genomics screening identifies modulators of neuronal activity.

Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders.

Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a ß-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.

Spironolactone is an antagonist of NRG1-ERBB4 signaling and schizophrenia-relevant endophenotypes in mice.

Enhanced NRG1-ERBB4 signaling is a risk pathway in schizophrenia, and corresponding mouse models display several endophenotypes of the disease. Nonetheless, pathway-directed treatment strategies with clinically applicable compounds have not been identified. Here, we applied a cell-based assay using the split TEV technology to screen a library of clinically applicable compounds to identify modulators of NRG1-ERBB4 signaling for repurposing. We recovered spironolactone, known as antagonist of corticosteroids, as an inhibitor of the ERBB4 receptor and tested it in pharmacological and biochemical assays to assess secondary compound actions. Transgenic mice overexpressing Nrg1 type III display cortical Erbb4 hyperphosphorylation, a condition observed in postmortem brains from schizophrenia patients. Spironolactone treatment reverted hyperphosphorylation of activated Erbb4 in these mice. In behavioral tests, spironolactone treatment of Nrg1 type III transgenic mice ameliorated schizophrenia-relevant behavioral endophenotypes, such as reduced sensorimotor gating, hyperactivity, and impaired working memory. Moreover, spironolactone increases spontaneous inhibitory postsynaptic currents in cortical slices supporting an ERBB4-mediated mode-of-action. Our findings suggest that spironolactone, a clinically safe drug, provides an opportunity for new treatment options for schizophrenia.

Characterizing Dynamic Protein-Protein Interactions Using the Genetically Encoded Split Biosensor Assay Technique Split TEV.

Dynamic protein-protein interactions (PPIs) are fundamental building blocks of cellular signaling and monitoring their regulation promotes the understanding of signaling in health and disease. Genetically encoded split protein biosensor assays, such as the split TEV method, have proved to be highly valuable when studying regulated PPIs in living cells. Split TEV is based on the functional complementation of two previously inactive TEV protease fragments fused to interacting proteins and provides a robust, sensitive and flexible readout to monitor PPIs both at the membrane and in the cytosol. Thus, split TEV can be used to analyze interactomes of receptors, membrane-associated proteins, and cytosolic proteins. In particular, split TEV is useful to assay activities of relevant drug targets, such as receptor tyrosine kinases and G protein-coupled receptors, in compound screens. As split TEV uses genetically encoded readouts, including standard reporters based on fluorescence and luminescence, the technique can also be combined with scalable molecular barcode reporter systems, allowing the integration into multiplexed high-throughput assay approaches. Split TEV can be used in standard heterologous cell lines and primary cell types, including neurons, either in a transient or stably integrated format. When using cell lines, the basic protocol takes 30-96 h to complete, depending on the complexity of the experimental question addressed.

Monitoring G protein-coupled receptor activation using the protein fragment complementation technique split TEV.

G protein-coupled receptors (GPCRs) modulate cellular signaling, often in a ligand-specific manner. Cellular effects regulated include differentiation, proliferation, hormonal regulation, and neuronal activity. Further, they are involved in many disease-relevant processes, such as cancer and neurodevelopmental diseases, and represent the largest class of drug targets. Therefore, monitoring how GPCRs are regulated in their activity is crucial to understand their role in physiological processes and implications for drug development. Split TEV, a method based on TEV protease fragment complementation, can be used to sensitively assay GPCR activities in living cells. The activity of a given GPCR is monitored through its binding to ß-arrestin. Split TEV reporters provide at minimum a two-step amplification process facilitating a flexible format and a robust readout. For the initial setup, a GPCR of interest and ß-arrestin are fused to the N- and C-terminal fragments of the TEV protease, and occurred interactions are indicated by increased fluorescence or luminescence of TEV cleavage-dependent reporters. The experimental procedure takes 24-72 h to complete, depending on the cell type and complexity of the experimental setup applied.

Evolutionary and molecular facts link the WWC protein family to Hippo signaling.

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family that also includes the highly similar proteins WWC2 and WWC3. Although the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. Here, we describe evolutionary, molecular, and functional aspects of the WWC family. We show that the WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapods, the three family members, KIBRA/WWC1, WWC2, and WWC3, are found, except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway.

Kibra is a regulator of the Salvador/Warts/Hippo signaling network.

The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. Here, we identify Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. We suggest that Kibra is part of an apical scaffold that promotes SWH pathway activity.

Split-cre complementation indicates coincident activity of different genes in vivo.

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive "split-Cre" fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.

Disturbed clockwork resetting in Sharp-1 and Sharp-2 single and double mutant mice.

BACKGROUND: The circadian system provides the basis to anticipate and cope with daily recurrent challenges to maintain the organisms' homeostasis. De-synchronization of circadian feedback oscillators in humans causes 'jet lag', likely contributes to sleep-, psychiatric-, metabolic disorders and even cancer. However, the molecular mechanisms leading to the disintegration of tissue-specific clocks are complex and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Based on their circadian expression and cell culture experiments, the basic Helix-Loop-Helix (bHLH) transcription factors SHARP-1(Dec2) and SHARP-2(Stra13/Dec1) were proposed as novel negative regulators of the molecular clock. To address their function in vivo, we generated Sharp-1 and Sharp-2 single and double mutant mice. Our experiments reveal critical roles for both factors in regulating period length, tissue-specific control of clock gene expression and entrainment to external cues. Light-pulse experiments and rapid delays of the light-dark cycle (experimental jet lag) unravel complementary functions for SHARP-1 and SHARP-2 in controlling activity phase resetting kinetics. Moreover, we show that SHARP-1 and 2 can serve dual functions as repressors and co-activators of mammalian clock gene expression in a context-specific manner. This correlates with increased amplitudes of Per2 expression in the cortex and liver and a decrease in the suprachiasmatic nucleus (SCN) of double mutant mice. CONCLUSIONS/SIGNIFICANCE: The existence of separate mechanisms regulating phase of entrainment, rhythm amplitude and period length has been postulated before. The differential effects of Sharp-deficiency on rhythmicity and behavioral re-entrainment, coupled to tissue-dependent regulatory functions, provide a new mechanistic basis to further understand the complex process of clock synchronizations.

Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV.

BACKGROUND: Regulated protein-protein interactions (PPIs) are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture. RESULTS: In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3epsilon and zeta in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We furthermore applied split-TEV assays to measure the phosphorylation-dependent interactions of Neuregulin-1-stimulated ErbB4 receptors with several adapter proteins. CONCLUSION: Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically at the membrane and in the cytosol of heterologous and primary cultured mammalian cells. Given the high sensitivity of the split-TEV system, all assays were performed in multi-plate formats and could be adapted for higher throughput to screen for pharmacologically active substances.