Biology subjects

Tsvetanova, N. G.

Publications and source records attributed to Tsvetanova, N. G..

5 recordsLinked to original sources

bioRxiv2025

GPCR endocytosis rewires neuronal gene expression and cellular architecture

In the brain, G protein-coupled receptors (GPCRs) regulate neuronal excitability, synaptic transmission, and behavior by engaging transcriptional and translational programs that produce enduring changes in cellular function and architecture. However, the molecular mechanisms that couple GPCR activation to these adaptations remain poorly understood. Here, we demonstrate that the beta-adrenergic receptor ({beta}2AR), a mediator of noradrenaline in the central nervous system, remodels neuronal morphology through compartmentalized signaling pathways that orchestrate distinct layers of gene regulation. Following stimulation, {beta}2ARs remain active on endosomes, and their intracellular signaling promotes dendritic growth and synapse formation in human iPSC-derived and primary cortical neurons. These structural effects are driven by two coordinated regulatory axes: PKA/CREB-dependent transcription of morphogenesis-related genes and PKA/mTOR-dependent translation of components of the protein synthesis machinery. Altogether, this work defines novel spatial and biochemical principles by which GPCR signaling drives structural reorganization and functional adaptations in neurons.

cell biology↗

bioRxiv2024

Extensive location bias of the GPCR-dependent translatome via site-selective activation of mTOR

G protein-coupled receptors (GPCRs) modulate various physiological functions by re-wiring cellular gene expression in response to extracellular signals. Control of gene expression by GPCRs has been studied almost exclusively at the transcriptional level, neglecting an extensive amount of regulation that takes place translationally. Hence, little is known about the nature and mechanisms of gene-specific post-transcriptional regulation downstream of receptor activation. Here, we apply an unbiased multiomics approach to delineate an extensive translational regulatory program initiated by the prototypical beta2-adrenergic receptor ({beta}2-AR) and provide mechanistic insights into how these processes are orchestrated. Using ribosome profiling (Ribo-seq), we identify nearly 120 novel gene targets of adrenergic receptor activity which expression is exclusively regulated at the level of translation. We next show that all translational changes are induced selectively by endosomal {beta}2-ARs. We further report that this proceeds through activation of the mammalian target of rapamycin (mTOR) pathway. Specifically, within the set of translational GPCR targets we discover significant enrichment of genes with 5 terminal oligopyrimidine (TOP) motifs, a gene class classically known to be translationally regulated by mTOR. We then demonstrate that endosomal {beta}2-ARs are required for mTOR activation and subsequent mTOR-dependent TOP mRNA translation. Together, this comprehensive analysis of drug-induced translational regulation establishes a critical role for location-biased GPCR signaling in fine-tuning the cellular protein landscape.

cell biology↗

bioRxiv2023

The psychosis risk factor RBM12 encodes a novel repressor of GPCR/cAMP signal transduction

RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293 cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cyclic AMP/protein kinase A (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR/cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.

cell biology↗

bioRxiv2021

Endosomal cAMP production broadly impacts the cellular phosphoproteome

Endosomal signaling downstream of G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, highconfidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that the same amount of cAMP produced from endosomes led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56{delta} as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.

cell biology↗

bioRxiv2020

A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and weaker phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with weaker phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators. Author summaryCells sense and respond to changes in their surrounding environment through G protein-coupled receptors (GPCRs) and their associated cascades. The proper function of these pathways is essential to human physiology, and GPCRs have become a prime target for drug development for a range of human diseases. Therefore, it is of utmost importance to be able to map how these pathways operate to enable cells to fine-tune their responsiveness. Here, we describe a screening approach that we have devised to systematically identify regulators of GPCR function. We have developed a sortable reporter system and coupled that with silencing of genes across the entire human genome in order to uncover a range of novel mediators of GPCR activity. We characterize a few of these new regulators and show that they function at different steps of the cascade. Therefore, this study serves as proof of principle for the new screening platform. We envision that the approach can be used to dissect additional dimensions of GPCR function, including regulators of drug-specific responses, functional characterization of receptor features, and identification of novel drugs, and thus advance a genome-scale understanding of these critical pathways.

cell biology↗