Protein-protein Interactions In Cells Research Articles

Mapping protein-protein interactions by mass spectrometry.

Protein-protein interactions (PPIs) are essential for numerous biological activities, including signal transduction, transcription control, and metabolism. They play a pivotal role in the organization and function of the proteome, and their perturbation is associated with various diseases, such as cancer, neurodegeneration, and infectious diseases. Recent advances in mass spectrometry (MS)-based protein interactomics have significantly expanded our understanding of the PPIs in cells, with techniques that continue to improve in terms of sensitivity, and specificity providing new opportunities for the study of PPIs in diverse biological systems. These techniques differ depending on the type of interaction being studied, with each approach having its set of advantages, disadvantages, and applicability. This review highlights recent advances in enrichment methodologies for interactomes before MS analysis and compares their unique features and specifications. It emphasizes prospects for further improvement and their potential applications in advancing our knowledge of PPIs in various biological contexts.

Light-activated BioID - an optically activated proximity labeling system to study protein-protein interactions.

Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called light-activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate, in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID and initiate biotinylation. Turning off the light leads to the dissociation of CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin-binding partners with higher accuracy and significantly fewer false positives than TurboID.

Abstract P2-11-35: A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer

Abstract A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer Han Gao1, Fiona Ruge1, Lin Ye1, Jane Lane1, Eleri Davies2, Wen G. Jiang1, Tracey A. Martin1 1Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK 2Wales Breast Centre, University Llandough Hospital, Cardiff CF64 2XX, UK Introduction. MUPP1 (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) or MPDZ is a scaffold protein with multiple PDZ domains, critical for multiple protein docking and protein-protein interactions in cells. MUPP1 is particularly interesting as it offers docking sites for a number of tight junction (TJ) proteins and proteins known to regulate cell junction and motility, including angiomotin (AMOT) and angiomotin-like proteins, the latter being important in regulating cell migration of endothelial cells and an important member in TJ protein complexes. The MUPP1 protein complex at cell junctions also includes CAR/CXADR (coxsackie adenovirus receptor) and members of the ERM (Moesin-Ezrin-Radixin) protein family. It was proposed that the junctional-related MUPP1 docking protein complex may collectively have a role in the development and progression of cancer including breast cancer. The present study examined the profile and integrated expression of these MUPP1 docking molecules in relationship with the outcome, clinical and pathological factors of breast cancer. Methods. Using the datasets of an existing breast cancer cohort at Cardiff, we visited the expression profile of the MUPP1/MPDZ docking molecules including MUPP1, four members of the ERM family, including ezrin, moesin, radixin and EHM2 (also known as NF2) and CAR/CXADR (coxsackie adenovirus receptor). The profile and integrated expression of the profile were analysed against clinical, pathological and outcome factors and also the hormone receptor status of breast cancers. Results. MUPP1 is a weakly linked to the disease progression, so as angiomotin, angiomotin-like1 and angiomotin-like2. Alone, only EHM2 showed significance in linking with the survival of the patient’s OS (p=0.38), neither of the remaining MUPP1 complex molecules, including MUPP1, AMOT, AMOTL1, AMOTL2, ezrin, moesin and radixin was able to offer a significant predictive value for the survival of the patients. However, it was found that MUPP1 has a significant correlation with AMOTs, EHM2 and ezrin. When these significantly correlated molecules were integrated in the analysis, the integrated expression pattern showed a highly significant value in evaluating the overall survival (p=0.003) and disease-free survival (p=0.007). Both of these predictive powers are independent in a multivariate analyses including the clinical and hormone receptors for OS (p=0.022, Hazard Ratio (HR)=1.352 95%CI (1.044-1.752)) and RFS (p=0.023, HR=1.241 (95% CI. 1.031-1.498)). It was interesting to note that the link between the expression pattern and OS was more prominent in ER negative (p=0.018) than in ER positive tumours (p=0.177). The same was observed with disease free survival for ER negative (p=0.011) compared with ER positive (p=0.462) tumours. Non-triple negative breast cancer had shown a similar sensitivity compared with triple negative breast cancer. Discussion. MUPP1, a PDZ domain containing docking protein closed linked with TJ structure, forms an interesting expression profile with its docking molecules including angiomotins and EHM2 that allows evaluation of disease progress and clinical outcome of the patients with breast cancer. Citation Format: Han Gao, Fiona Ruge, Lin Ye, Jane Lane, Eleri Davies, Wen G. Jiang, Tracey A. Martin. A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-35.

In-cellulo chemical cross-linking to visualize protein-protein interactions.

Reversible protein-protein interaction in cells is an integral and central aspect of intracellular signaling mechanisms. This allows distinct signaling cascades to become active upon stimulation with external signal resulting in cellular and physiological responses. Several distinct methods are currently available and utilized routinely to monitor protein-protein interactions including co-immunoprecipitation (co-IP). An inherent limitation associated with co-IP assay however is the inability to efficiently capture transient and short-lived interactions in cells. Chemical cross-linking of such transient interactions in cellular context using cell permeable reagents followed by co-IP overcomes this limitation, and allows a simplified approach without requiring any sophisticated instrumentation. In this chapter, we present a step-by-step protocol for monitoring protein-protein interaction by combining chemical cross-linking and co-immunoprecipitation using GPCR-β-arrestin complex as a case example. This protocol is based on previously validated method that can potentially be adapted to capture and visualize transient protein-protein interactions in general.

Proximity Ligation in Situ Assay to Monitor Autophagy-Related Protein Interactions and Autophagy in Cancer Cells.

Proximity ligation in situ assay (PLISA) is a powerful method to quantify endogen protein-protein interactions in cells and simultaneously identify localization of these interactions. PLISA can be used to quantify autophagy flux and can as well be adapted to assess global autophagy (SQSTM1/P62-LC3B interaction) or specific autophagy, such as mitophagy (NIX-LC3B). Here, we describe a step-by-step method to monitor autophagy using PLISA in adherent cancer cells.

Abstract P176: Discovery and development of novel covalent inhibitors of the YAP-TEAD transcription activity

Abstract TEAD (TEA domain transcription factor) is a transcription factor. As a major effector of the Hippo signaling pathway, TEAD is reported to be responsible for cell proliferation, apoptosis, and stem cell self-renewal. Emerging evidence has shown that TEADs play a prominent role in the development, progression, and metastasis of cancers such as mesothelioma, liposarcoma, pancreatic, etc. Small-molecule inhibitors of TEADs could be a novel targeted therapy for multiple cancers. Using its proprietary chemoproteomic platform IMTACTM (Isobaric Mass Tagged Affinity Characterization), BridGene has screened its unique covalent library against live-cell proteomes. Three distinct series of hits, with MCS Tanimoto Coefficients at ~0.3 between any two series, were identified for TEAD proteins with nanomolar affinity and high proteomic selectivity. The covalent interactions between the hit and TEAD proteins were confirmed using orthogonal fluorescence-based experiments. Mass Spec analysis and mutagenesis experiments identified the site of modification within the central lipid (palmitate) binding pocket of TEAD1. Multiple novel and potent small molecule inhibitors were obtained with good druggability. Several functional assays were established to measure inhibition of TEAD-YAP transcription activity/disruption including Luciferase assay in MCF7-Luc, CTG (cell-titer Glo), and ELISA. When evaluated in vitro, BridGene’s TEAD inhibitors exhibited anti-proliferative properties against NF2-deficient mesothelioma cell lines (NCI-H226, NF2-/-) with low nM IC50, but had little effects on in NF2 wildtype cancer cell lines (NCI-H2452, NF2+/+) or HepG2 cell line. In an ELISA assay, BridGene’s small molecules can potently disrupt TEAD-YAP protein-protein interaction in cells. In MCF7-TEAD1 luciferase reporter cells, compounds showed single-digit nM potency in disrupting TEAD downstream gene expression. BridGene is developing covalent TEAD inhibitors to treat multiple cancers that are dependent on TEAD activity, including mesothelioma, cholangiocarcinoma, liposarcoma, pancreatic cancer, etc. With their high potency and proteomic selectivity, these TEAD inhibitors can potentially act as single-agent therapy, or work in combination with other care agents to treat multiple cancers. Citation Format: Shirley Guo, Cindy Huang, C. Glenn Begley, Michael J. Bishop, Ping Cao. Discovery and development of novel covalent inhibitors of the YAP-TEAD transcription activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P176.

Compartmentalization-aided interaction screening reveals extensive high-order complexes within the SARS-CoV-2 proteome.

Bearing a relatively large single-stranded RNA genome in nature, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. In this study, we develop an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we map the interaction network among RTC-related viral proteins. We identify a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC leads to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient and robust interaction screening strategy, and they indicate the existence of a complex interaction network among RTC-related factors, thus opening up opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.

Abstract 2410: The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion

Abstract Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and has the worst prognosis of any breast cancer sub-type. More than 80% of TNBC patients harbor mutated TP53. Some mutant p53 proteins (mtp53) exhibit gain-of function (GOF) oncogenic characteristics, contributing to the decreased prognosis for TNBC patients, but also providing a potential therapeutic target for this aggressive disease. Our group previously found mtp53 R273H interacts with replicating DNA, and increases chromatin-bound replication proteins MCM2-7, PCNA, and PARP1. Mutant p53 retains an intact C-terminal domain (CTD), the domain which facilitates non-specific DNA binding. We hypothesize that the CTD of mtp53 facilitates increased DNA replication processivity. Methods: To test this hypothesis, we have transiently transfected human cells to compare wild-type p53, and R273H mtp53 full-length and CTD-truncated variants. We used CRISPR/Cas9 to generate endogenous CTD truncations of mtp53 R273H in the breast cancer cell line MDA-MB-468. Specifically, clones were used which have mtp53 CTD truncations (R273HΔ381-388 and R273HΔ347-393) and one clone with a frameshift resulting in no detectable mtp53 protein (R273Hfs387). Various western blotting techniques were used to assess protein level and sub-cellular localization, including the iPOND (isolation of proteins on nascent DNA) assay. Several microscopy techniques were used to visualize protein-protein interactions in cells including immunofluorescence, proximity ligation, and DNA fiber assays. Results: Our data show mtp53 R273H and wild-type p53 proteins with CTD truncations tether less to chromatin than their full-length counterparts. Also, our data shows that the truncated R273HΔ381-388 mtp53 is in close proximity to PARylated proteins (and potentially even PARylated itself), but the R273HΔ347-393 truncation is not in close proximity to PARylated proteins. Additionally, R273HΔ381-388 and R273HΔ347-393 demonstrate delayed cell cycle progression and reduced proliferation compared to MDA-MB-468 cells. We observe DNA replication processivity is decreased in 468 R273HΔ381-388 and R273Hfs387 cells. Processivity, but not proliferation rate, is rescued in 468 R273HΔ347-393 cells. Conclusions: The combination of reduced cell proliferation in all CTD-truncated cells, and reduced DNA replication processivity in R273HΔ381-388 and R273Hfs387 cells, but not in R273HΔ347-393 indicates a nuanced role for the CTD in DNA replication and S-phase progression. Citation Format: Devon Lundine, Zafar Syed, Viola Ellison, George Annor, Gu Xiao, Jill Bargonetti. The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2410.

Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.

Proximity biotinylation by AirID to map protein-protein interactions in cells

Proximity biotinylation by AirID to map protein-protein interactions in cells

Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ.

Biochemical methods can reveal stable protein-protein interactions occurring within cells, but the ability to observe transient events and to visualize the subcellular localization of protein-protein interactions in cells and tissues in situ provides important additional information. The Proximity Ligation Assay® (PLA) offers the opportunity to visualize the subcellular location of such interactions at endogenous protein levels, provided that the probes that recognize the target proteins are within 40 nm. This sensitive technique not only elucidates protein-protein interactions, but also can reveal post-translational protein modifications. The technique is useful even in cases where material is limited, such as when paraffin-embedded clinical specimens are the only available material, as well as after experimental intervention in 2D and 3D model systems. Here we describe the basic protocol for using the commercially available Proximity Ligation Assay™ materials (Sigma-Aldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Proximity ligation assay Support Protocol 1: Antigen retrieval method for formalin-fixed, paraffin-embedded tissues Support Protocol 2: Creation of custom PLA probes using the Duolink™ In Situ Probemaker Kit when commercially available probes are not suitable Basic Protocol 2: Imaging, quantification, and analysis of PLA signals.

Protein-Peptide Binding Energetics under Crowded Conditions.

Nearly all biological processes, including strictly regulated protein-protein interactions fundamental in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction is between a 7 kDa SH3 domain and a 25 kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its key role in the mitogen-activated protein kinase signaling pathway of all eukaryotes, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. Here, we investigate the effects of proteins, sugars, and urea, at high g/L concentrations, on the kinetics and equilibrium thermodynamics of binding between SH3 and two SOS-derived peptides using 19F NMR lineshape analysis. We also analyze the temperature dependence, which enables quantification of the enthalpic and entropic contributions. The energetics of SH3-peptide binding in proteins differs from those in the small molecules we used as control cosolutes, demonstrating the importance of using proteins as physiologically relevant cosolutes. Although most of the protein cosolutes destabilize the SH3-peptide complexes, the effects are nongeneralizable and there are subtle differences, which are likely from weak nonspecific interactions between the test proteins and the protein crowders. We also quantify the effects of cosolutes on SH3 translational and rotational diffusion to rationalize the effects on association rate constants. The absence of a correlation between the SH3 diffusion data and the kinetic data in certain cosolutes suggests that the properties of the peptide in crowded conditions must be considered when interpreting energetic effects. These studies have implications for understanding protein-protein interactions in cells and show the importance of using physiologically relevant cosolutes for investigating macromolecular crowding effects.

Visualizing the in vitro assembly of tropomyosin/actin filaments using TIRF microscopy.

Tropomyosins are elongated alpha-helical proteins that form co-polymers with most actin filaments within a cell and play important roles in the structural and functional diversification of the actin cytoskeleton. How the assembly of tropomyosins along an actin filament is regulated and the kinetics of tropomyosin association with an actin filament is yet to be fully determined. A recent series of publications have used total internal reflection fluorescence (TIRF) microscopy in combination with advanced surface and protein chemistry to visualise the molecular assembly of actin/tropomyosin filaments in vitro. Here, we review the use of the in vitro TIRF assay in the determination of kinetic data on tropomyosin filament assembly. This sophisticated approach has enabled generation of real-time single-molecule data to fill the gap between in vitro bulk assays and in vivo assays of tropomyosin function. The in vitro TIRF assays provide a new foundation for future studies involving multiple actin-binding proteins that will more accurately reflect the physiological protein-protein interactions in cells.

Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM

Fluorescence lifetime imaging (FLIM) is a quantitative, intensity-independent microscopical method for measurement of diverse biochemical and physical properties in cell biology. It is a highly effective method for measurements of Förster resonance energy transfer (FRET), and for quantification of protein-protein interactions in cells. Time-domain FLIM-FRET measurements of these dynamic interactions are particularly challenging, since the technique requires excellent photon statistics to derive experimental parameters from the complex decay kinetics often observed from fluorophores in living cells. Here we present a new time-domain multi-confocal FLIM instrument with an array of 64 visible beamlets to achieve parallelised excitation and detection with average excitation powers of ~ 1–2 μW per beamlet. We exemplify this instrument with up to 0.5 frames per second time-lapse FLIM measurements of cAMP levels using an Epac-based fluorescent biosensor in live HeLa cells with nanometer spatial and picosecond temporal resolution. We demonstrate the use of time-dependent phasor plots to determine parameterisation for multi-exponential decay fitting to monitor the fractional contribution of the activated conformation of the biosensor. Our parallelised confocal approach avoids having to compromise on speed, noise, accuracy in lifetime measurements and provides powerful means to quantify biochemical dynamics in living cells.

Validation of the Applicability of In-Cell Fast Photochemical Oxidation of Proteins across Multiple Eukaryotic Cell Lines.

Fast photochemical oxidation of proteins (FPOP), a hydroxyl radical-based protein footprinting method, coupled to mass spectrometry has been extensively used to study protein structure and protein-protein interactions in vitro. This method utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids and has recently been demonstrated to modify proteins within live cells (IC-FPOP) and Caenorhabditis elegans. Here, we have expanded the application of IC-FPOP into a variety of commonly used cell lines to verify the applicability of the method across various cellular systems. IC-FPOP was able to successfully modify proteins in five different cell lines (Vero, HEK 293T, CHO, MCF-10A, and MCF-7). To increase the number of oxidatively modified proteins identified, we have also employed the use of offline high pH reversed-phase liquid chromatography (RPLC) followed by concatenation and online low-pH RPLC. The coupling of IC-FPOP to 2D-LC MS/MS resulted in a 1.7-fold increase in total identifications of oxidatively modified proteins, which expanded the dynamic range of the method. This work demonstrates the efficacy of using IC-FPOP to study protein-protein interactions in cells.

Split T7 RNA polymerase biosensors to study multiprotein interaction dynamics.

Protein-protein interactions (PPIs) are involved in nearly all cellular processes. PPIs are particularly crucial for mediating selectivity along signaling pathways. Thus, measuring the competitive interplay between PPIs in a cell is important for both understanding fundamental cellular regulation and developing therapeutics targeting those whose dysregulation is associated with disease. A variety of split protein reporter-based tools are available to measure if two proteins interact within a cell and thereby characterize the general determinants of their interactions. PPIs, however, occur within complex networks facilitated by dynamic biophysical nuances that determine activity and selectivity. Evolved, proximity-dependent split T7 RNA polymerase (RNAP) biosensors have recently been used to perform deep mutational scanning of PPI interfaces, and to create synthetic gene circuits. In this chapter, we present the application of proximity-dependent split RNAP biosensors as a method to measure multidimensional PPIs in live cells. Orthogonal split RNAP "tags" encode each interaction in a unique RNA signal, thereby enabling the study of multiple competitive PPIs in live cells. Each unique RNA signal can be quantified via established RNA analysis methods. Herein, we provide advice and protocols to aid other researchers in using the split RNAP biosensor, focusing primarily on how to detect multiple PPIs in mammalian cells, including their dynamic interplay in the presence of small molecule inhibitors.

Photoproximity Profiling of Protein-Protein Interactions in Cells.

We report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction- and light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled unbiased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of PhotoPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD, and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic subcellular and protein interaction "social network" of a redox-sensitive protein in cells with high temporal resolution.

Genetic code expansion and photocross-linking identify different β-arrestin binding modes to the angiotensin II type 1 receptor

The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein-coupled receptor (GPCR) family and binds β-arrestins (β-arrs), which regulate AT1R signaling and trafficking. These processes can be biased by different ligands or mutations in the AGTR1 gene. As for many GPCRs, the exact details for AT1R-β-arr interactions driven by AngII or β-arr-biased ligands remain largely unknown. Here, we used the amber-suppression technology to site-specifically introduce the unnatural amino acid (UAA) p-azido-l-phenylalanine (azF) into the intracellular loops (ICLs) and the C-tail of AT1R. Our goal was to generate competent photoreactive receptors that can be cross-linked to β-arrs in cells. We performed UV-mediated photolysis of 25 different azF-labeled AT1Rs to cross-link β-arr1 to AngII-bound receptors, enabling us to map important contact sites in the C-tail and in the ICL2 and ICL3 of the receptor. The extent of AT1R-β-arr1 cross-linking among azF-labeled receptors differed, revealing variability in β-arr's contact mode with the different AT1R domains. Moreover, the signature of ligated AT1R-β-arr complexes from a subset of azF-labeled receptors also differed between AngII and β-arr-biased ligand stimulation of receptors and between azF-labeled AT1R bearing and that lacking a bias signaling mutation. These observations further implied distinct interaction modalities of the AT1R-β-arr1 complex in biased signaling conditions. Our findings demonstrate that this photocross-linking approach is useful for understanding GPCR-β-arr complexes in different activation states and could be extended to study other protein-protein interactions in cells.

Detecting In-Situ oligomerization of engineered STIM1 proteins by diffraction-limited optical imaging.

Several signaling proteins require self-association of individual monomer units to be activated for triggering downstream signaling cascades in cells. Methods that allow visualizing their underlying molecular mechanisms will immensely benefit cell biology. Using enhanced Green Fluorescent Protein (eGFP) complementation, here I present a functional imaging approach for visualizing the protein-protein interaction in cells. Activation mechanism of an ER (endoplasmic reticulum) resident Ca2+ sensor, STIM1 (Stromal Interaction Molecule 1) that regulates store-operated Ca2+ entry in cells is considered as a model system. Co-expression of engineered full-length human STIM1 (ehSTIM1) with N-terminal complementary split eGFP pairs in mammalian cells fluoresces to form ‘puncta’ upon a drop in ER lumen Ca2+ concentration. Quantization of discrete fluorescent intensities of ehSTIM1 molecules at a diffraction-limited resolution revealed a diverse set of intensity levels not exceeding six-fold. Detailed screening of the ehSTIM1 molecular entities characterized by one to six fluorescent emitters across various in-plane sections shows a greater probability of occurrence for entities with six emitters in the vicinity of the plasma membrane (PM) than at the interior sections. However, the number density of entities with six emitters was lesser than that of others localized close to the PM. This finding led to hypothesize that activated ehSTIM1 dimers perhaps oligomerize in bundles ranging from 1–6 with an increased propensity for the occurrence of hexamers of ehSTIM1 dimer units close to PM even when its partner protein, ORAI1 (PM resident Ca2+ channel) is not sufficiently over-expressed in cells. The experimental data presented here provide direct evidence for luminal domain association of ehSTIM1 monomer units to trigger activation and allow enumerating various oligomers of ehSTIM1 in cells.

Combining TIR and FRET in Molecular Test Systems.

Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.