Endocytosis sculpts distinct cAMP signal transduction by endogenously coexpressed GPCRs
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ABSTRACT:
PROVIDER: PRJNA1225749 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32401372.fastq.gz | Fastqsanger.gz | |||
| SRR32401373.fastq.gz | Fastqsanger.gz | |||
| SRR32401374.fastq.gz | Fastqsanger.gz | |||
| SRR32401375.fastq.gz | Fastqsanger.gz | |||
| SRR32401376.fastq.gz | Fastqsanger.gz |
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Project description:Many G protein-coupled receptors (GPCRs) trigger a second phase of G protein-dependent signaling from internal membranes after agonist-induced endocytosis. However, individual GPCRs differ significantly in their ability to internalize after activation, and it remains unclear if this confers selectivity on cellular signaling through natively coexpressed GPCRs. We addressed this question by examining the activation of the cyclic AMP (cAMP) / cAMP-dependent protein kinase (PKA) pathway by three ligands that stimulate three distinct, endogenously coexpressed GPCRs in HEK293 cells: isoproterenol (Iso) which primarily activates the β2-adrenergic receptor (β2AR), vasoactive intestinal peptide (VIP) which primarily activates the vasoactive intestinal peptide receptor 1 (VIPR1/VPAC1), and 5'-N-ethylcarboxamidoadenosine (NECA) which primarily activates the adenosine 2B receptor (A2BR). Using location-targeted biosensors and a transcriptional reporter, we demonstrate that each ligand triggers a unique cellular signaling profile and that these responses are differentially sensitive to endocytic inhibition. VIP elicited a response that was endocytosis-dependent at every level in the pathway tested, from upstream global cAMP elevation to downstream activation of nuclear PKA, while Iso elicited a response that was dependent on endocytosis selectively at downstream steps. In contrast, NECA robustly activated the entire cAMP signaling cascade independently of endocytosis, consistent with our observation that human A2BR does not robustly internalize after activation. We conclude that endocytosis indeed sculpts downstream cAMP signaling by GPCRs in a receptor-specific manner. Our results add to the evolving view of compartmentalized signaling in the cAMP / PKA pathway and suggest that differences in GPCR trafficking can encode receptor-specific signaling profiles through a shared signal transduction pathway.
2025-02-24 | GSE290019 | GEO
Project description:G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization. Apart from its temporal impact through prolonging the cellular response, we wondered if the endosome-initiated signal encodes any discrete spatial information. Using the beta2-adrenoceptor (β2-AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP-dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis. These findings reveal a discrete principle for achieving cellular signalling specificity, based on endosome-mediated spatial encoding of intracellular second messenger production and 'location aware' downstream transcriptional control.
2014-11-01 | GSE57274 | GEO
Project description:Purpose: The goals of this study are to identify Gs-linked GPCRs that are endogenously expressed by mouse adipose tissue, we subjected RNA prepared from isolated mouse adipocytes (iWAT and eWAT) and BAT tissue to RNA-seq analysis. Methods: Total RNA extracted from mature adipocytes (iWAT and eWAT) and BAT tissue of C57BL/6J mice (16-week old males) maintained on regular chow were used to construct high throughput sequencing libraries. RNAs with RIN >8 (assessed by the Agilent 2100 Bioanalyzer system) were used to prepare transcriptome libraries using the NEBNext Ultra RNA library prep kit (New England Biolabs). High throughput RNA-sequencing was performed using a HiSeq 2500 instrument (Illumina) at the NIDDK Genomic Core Facility (NIH, Bethesda, MD). Raw reads were mapped to the mouse (mm9) genome. GPCRs were extracted from the RNA-seq data using R form. Gs-coupled GPCRs were identified using the IUPHAR/BPS Guide to Pharmacology Database (https://www.guidetopharmacology.org/). Results: Our study demonstrated that mouse adipocytes/BAT tissue express several GPCRs that are selectively coupled to Gs, including the V2 vasopressin receptor, the glucagon receptor, and different melanocortin receptor subtypes.
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Project description:Purpose: The goals of this study are to identify GPCRs that are endogenously expressed by high fat diet fed mouse adipose tissue, we subjected RNA prepared from isolated mouse adipocytes (iWAT and eWAT) and BAT tissue to RNA-seq analysis. Methods: Total RNA extracted from mature adipocytes (iWAT and eWAT) and BAT tissue of C57BL/6 mice maintained on high fat diet for 12 weeks were used to construct high throughput sequencing libraries. RNAs with RIN >8 (assessed by the Agilent 2100 Bioanalyzer system) were used to prepare transcriptome libraries using the NEBNext Ultra RNA library prep kit (New England Biolabs). High throughput RNA-sequencing was performed using a Illumina HiSeq 2500 at the NIDDK Genomic Core Facility (NIH, Bethesda, MD). Raw reads were mapped to the mouse (mm9) genome. Results: Our study demonstrated that mouse adipocytes/BAT tissue from HFD-fed mice express several GPCRs.
2019-07-26 | GSE134914 | GEO
Project description:A deeper understanding of the regulation of G protein-coupled receptors (GPCRs) and their associated downstream cascades will provide critical insights into how these pathways shape human physiology and disease, and could yield novel therapeutic targets. Here, we validate and characterize RNA-binding motif 12 (Rbm12) as a repressor of GPCR/cAMP signaling. We established Rbm12 CRISPR KO HEK293 cells using two independent gRNAs and human iPSC-derived neurons (iNeuron) depleted of Rbm12 via CRISPR interference. Then, we performed basal gene expression profiling (HEK293) and basal + beta-2-adrenergic receptor induced (1 hour of 1 uM Isoproterenol) conditions (neurons).
2023-08-02 | GSE219195 | GEO
Project description:Hypoxia signature in Clear cell RCC A set of clear cell renal cell carcinomas were examined for the deregulation of hypoxia inducible and endocytosis regulated genes Tumor samples and Normal renal tissue GSE14762.rda contains an R data object
2009-02-09 | E-GEOD-14762 | biostudies-arrayexpress
Project description:Analysis of BMP-2 induced gene expression of C2C12 cells treated with the endocytosis inhibitor dynasore to identify gene expression profiles which are sensitive to endocytosis inhibition. Our data highlight the importance of endocytosis for transmission of an extracellular BMP-2 signal into the nucleus to activate a transcriptional gene network Total RNA obtained from C2C12 cells stimulated with BMP-2 together with the endocytosis inhibitor dynasore or DMSO as control compared to unstimulated cells treated with dynasore or DMSO alone.
2012-02-16 | E-GEOD-29373 | biostudies-arrayexpress
Project description:Targeting class A GPCRs for hard tissue regeneration
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Project description:Hypoxia signature in Clear cell RCC A set of clear cell renal cell carcinomas were examined for the deregulation of hypoxia inducible and endocytosis regulated genes
2009-02-10 | GSE14762 | GEO
Project description:Spinal muscular atrophy (SMA) is a common genetic motor neuron (MN) disease caused by low levels of the ubiquitously expressed housekeeping survival motor neuron (SMN) protein, whereas concomitant overexpression of plastin 3 (PLS3) protects from SMA. Here we identify neurocalcin delta (NCALD), a neuronal calcium sensor, as a second fully protective SMA modifier, which counteracts SMA pathology when downregulated. We show reduced Ca2+ influx and impaired endocytosis in SMA, which are crucial processes in synaptic vesicle recycling and neurotransmission. Remarkably, both reduced NCALD or increased PLS3 restore endocytosis in cell culture and MN function across species in zebrafish, worm and mouse SMA models, whereas Ca2+ influx remains disturbed. Furthermore, NCALD physically binds clathrin in a Ca2+-dependent manner. We propose that NCALD acts as a Ca2+-regulated inhibitor of endocytosis, and that endocytosis is critically affected in SMA. This finding opens new therapeutic avenues for SMA. Multifactorial design comparing affected patients and unaffected disease carriers against severly affected control group to identify disease modifying transcripts
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