Dataset Information

Orthogonal Peptide-Templated Labeling Elucidates Lateral ET_A R/ET_B R Proximity and Reveals Altered Downstream Signaling.

ABSTRACT: Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ET_A R and ET_B R), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ET_A R/ET_B R. Further, we demonstrate, that ET_A R expression regulates the signaling of co-expressed ET_B R. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

SUBMITTER: Wolf P

PROVIDER: S-EPMC9298254 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Orthogonal Peptide-Templated Labeling Elucidates Lateral ET<sub>A</sub> R/ET<sub>B</sub> R Proximity and Reveals Altered Downstream Signaling.

Wolf Philipp P Mohr Alexander A Gavins Georgina G Behr Victoria V Mörl Karin K Seitz Oliver O Beck-Sickinger Annette G AG

Chembiochem : a European journal of chemical biology 20211026 6

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this appr ...[more]

PMID: 34699123

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Project description:The endothelin system plays an important role in mediating vascular function. The endothelin-B receptor (ETBR) on endothelial cells mediates vasodilation via nitric oxide production. The vasodilatory effect of the ETBR is lost following menopause and may contribute to impaired vascular endothelial function in postmenopausal women (PMW). However, it is unclear if these functional changes are due to changes in ETBR expression on the endothelium. Therefore, the purpose of this study was to test the hypothesis that endothelial cell ETBR expression is lower in PMW compared with young women (YW). Primary endothelial cells were harvested from the antecubital vein of healthy PMW (n = 15, 60 ± 6 yr) and YW (n = 15, 22 ± 2 yr). Cells were identified as endothelial cells by staining for vascular endothelial cadherin, and nuclear integrity was assessed using 4',6-diamidino-2-phenylindole (DAPI). Within those cells, ETBR was quantified using immunocytochemistry; fluorescence intensity was measured in 30 cells and averaged for each participant. Endothelial function was assessed using brachial artery flow-mediated dilation (FMD). Endothelial cell ETBR expression was lower in PMW [0.46 ± 0.11 arbitrary units (AU)] compared with YW (0.58 ± 0.14 AU; P = 0.02). Furthermore, significant correlations between ETBR expression and FMD (r = 0.47, P < 0.01), total cholesterol (r = -0.38, P = 0.04), and LDL cholesterol (r = -0.39, P = 0.03) were observed. These data demonstrate that endothelial cell ETBR expression is attenuated in PMW. These novel findings provide additional insight into the mechanisms underlying vascular endothelial dysfunction in PMW.NEW & NOTEWORTHY Our study provides novel data demonstrating attenuated endothelial ETBR expression in postmenopausal women. Furthermore, our data extend current knowledge by demonstrating a positive relation between ETBR expression and brachial artery flow-mediated dilation. These findings provide additional mechanistic insight into vascular endothelial dysfunction in postmenopausal women.

Project description:Proximity labeling can be defined as an enzymatic "in-cell" chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not "accessible" via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell-cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in "spatiomics," that is, for the extraction of spatially distributed biological information in a cell or organism.Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This "in-cell" reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models.Simultaneously, chemists have also adopted the concept and employed chemical "photocatalysts" as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.

Dataset Information

Orthogonal Peptide-Templated Labeling Elucidates Lateral ET_A R/ET_B R Proximity and Reveals Altered Downstream Signaling.

Publications

Orthogonal Peptide-Templated Labeling Elucidates Lateral ET<sub>A</sub> R/ET<sub>B</sub> R Proximity and Reveals Altered Downstream Signaling.

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Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Publications

Orthogonal Peptide-Templated Labeling Elucidates Lateral ET<sub>A</sub> R/ET<sub>B</sub> R Proximity and Reveals Altered Downstream Signaling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Orthogonal Peptide-Templated Labeling Elucidates Lateral ET_A R/ET_B R Proximity and Reveals Altered Downstream Signaling.