β-Arrestins are key regulators and signal transducers of G protein–coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity ...and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase–phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine–threonine clusters in the receptor’s C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor–β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.
Despite decades of intensive search for compounds that modulate the activity of particular protein targets, a large proportion of the human kinome remains as yet undrugged. Effective approaches are ...therefore required to map the massive space of unexplored compound-kinase interactions for novel and potent activities. Here, we carry out a crowdsourced benchmarking of predictive algorithms for kinase inhibitor potencies across multiple kinase families tested on unpublished bioactivity data. We find the top-performing predictions are based on various models, including kernel learning, gradient boosting and deep learning, and their ensemble leads to a predictive accuracy exceeding that of single-dose kinase activity assays. We design experiments based on the model predictions and identify unexpected activities even for under-studied kinases, thereby accelerating experimental mapping efforts. The open-source prediction algorithms together with the bioactivities between 95 compounds and 295 kinases provide a resource for benchmarking prediction algorithms and for extending the druggable kinome.
Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol ...(25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that
is significantly upregulated in response to AngII stimulus. The
mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced
upregulation is type 1 angiotensin II receptor- and G
activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of
. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced
upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.
Background
Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and ...psoriatic arthritis (PsA) are associated with increased activity of osteoclasts.
Objectives
Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA.
Methods
Blood samples of healthy donors, RA, and PsA patients were collected, and monocytes were isolated and differentiated into osteoclasts
in vitro
using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA).
Results
The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts, expression of the proteins involved in metabolic activity, secretory function, and cell polarity is increased; by contrast, signaling pathways involved in the immune functions are downregulated. Interestingly, the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition, both in RA and PsA the differentiation is characterized by decreased metabolic activity, associated with various immune pathway activities; furthermore by accelerated cytokine production in RA.
Conclusions
Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA, which reveal important pathophysiological insights in both diseases.
(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid ...bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.
Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer–based assays are powerful approaches to achieve ...this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.
β-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G ...protein or β-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB
2
cannabinoid receptor (CB
2
R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB
2
R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB
2
receptors to G proteins and β-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased β-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB
2
receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB
2
R as a target for further development of biased receptor activation strategies.
The binding and function of β-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids ...responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established β-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with β-arrestins can be identified, a pattern that we name “arreSTick.” Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and β-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by β-arrestin2. Our findings unveil a broader role for β-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.
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•ArreSTick is a sequence pattern characteristic of stable GPCR-β-arrestin interaction•Numerous other proteins possess the arreSTick pattern•β-arrestin2 regulates HTSF1 via binding to its arreSTick motif
Tóth et al. developed a machine learning-based approach to identify sequence motifs in GPCRs, termed arreSTick, that mediate stable β-arrestin binding. They found that arreSTick is also present in many other proteins, suggesting that β-arrestins may regulate non-GPCR proteins through a phosphorylation-dependent mechanism, similar to its function in GPCRs.
The urine concentrating function of the kidney is essential to maintain the water homeostasis of the human body. It is mainly regulated by the arginine-vasopressin (AVP), which targets the type 2 ...vasopressin receptor (V2R) in the kidney. The inability of V2R to respond to AVP stimulation leads to decreased urine concentration and congenital nephrogenic diabetes insipidus (NDI). NDI is characterized by polyuria, polydipsia, and hyposthenuria. In this study, we identified a point mutation (S127F) in the
gene of an NDI patient, and we characterized the impaired function of the V2R mutant in HEK293 cells. Based on our data, the S127F-V2R mutant is almost exclusively located intracellularly in the endoplasmic reticulum (ER), and very few receptors were detected at the cell surface, where the receptor can bind to AVP. The overexpressed S127F-V2R mutant receptor has negligible cAMP generation capability compared to the wild-type receptor in response to AVP stimulation. Since certain misfolded mutant proteins, that are retained in the ER, can be rescued by pharmacological chaperones, we examined the potential rescue effects of two pharmacochaperones on the S127F-V2R. We found that pretreatment with both tolvaptan (an established V2R inverse agonist) and MCF14 compound (a cell-permeable high-affinity agonist for the V2R) were capable of partially restoring the cAMP generating function of the receptor in response to vasopressin stimulation. According to our data, both cell permeant agonists and antagonists can function as pharmacochaperones, and serve as the starting compounds to develop medicines for patients carrying the S127F mutation.
The CB1 cannabinoid receptor (CB1R) is the major cannabinoid receptor in neuronal cells and the brain, but it also occurs in endocrine cells and other peripheral tissues. CB1R is a member of the ...superfamily of G-protein-coupled receptors (GPCRs), which are characterized by seven transmembrane helices. The major mediators of CB1R are the G proteins of the Gi/o family, which inhibit adenylyl cyclases in most tissues and cells, and regulate ion channels, including calcium and potassium ion channels. Regulation of ion channels is an important component of neurotransmission modulation by endogenous cannabinoid compounds released in response to depolarization and Ca2+-mobilizing hormones. However, evidence exists that CB1Rs can also stimulate adenylyl cyclase via Gs, induce receptor-mediated Ca2+ fluxes and stimulate phospholipases in some experimental models. Stimulation of CB1R also leads to phosphorylation and activation of mitogen-activated protein kinases (MAPK), such as p42/p44 MAPK, p38 MAPK and c-Jun N-terminal kinase, which can regulate nuclear transcription factors. Activated and phosphorylated CB1Rs also associate with β-arrestin molecules, which can induce the formation of signalling complexes and participate in the regulation of GPCR signalling. Recent data also suggest that CB1Rs can form homo- and heterodimers/oligomers, and the altered pharmacological properties of these receptor complexes may explain the pharmacological differences observed in various tissues.
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