Biology subjects

Devost, D.

Publications and source records attributed to Devost, D..

5 recordsLinked to original sources

bioRxiv2025

Gαq-stimulated gene expression is insensitive to Bromo extra terminal domain inhibitors in HEK 293 cells

Bromodomain and extraterminal domain (BET) family proteins are ubiquitous transcriptional co-activators that function broadly in gene expression programs associated with cellular differentiation, proliferation, and stress responses. Pharmacological inhibition of BET proteins with small molecules that disrupt bromodomain engagement with acetyllysine residues (such as JQ1), or that drive their degradation through the ubiquitin-proteasome system (such as dBET6), ameliorates pathological gene expression in a range of systems and shows promise as a potential therapeutic strategy. BET involvement in gene expression responses is not universal, and understanding of the cell-type and signaling pathway requirements that dictate BET dependence remains incomplete. We previously demonstrated that, in neonatal rat cardiomyocytes, GPCR-induced hypertrophy response depended strongly on the BET protein Brd4 when signaling was coupled to Gs, but not Gq. Here we tested whether Brd4 was differentially responsive to G protein isoforms in HEK 293 cells by expressing Gs or Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Gq induced expression of a group of early response genes and inflammatory genes in a manner largely insensitive to pharmacological BET inhibition, consistent with our previous data in cardiomyocytes. Gs activated a small subset of the Gq-induced genes, but this effect was largely reversed by the BET degrader molecule dBET6. Our data further suggest that there may be general signaling requirements to activate Brd4 across cell types. Highlights- We tested whether Bromodomain and extraterminal domain (BET) family proteins were differentially responsive to G protein isoforms in HEK 293 cells by expressing Gs or Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). - Gq induced expression of a group of genes but was largely insensitive to pharmacological BET inhibition, consistent with our previous data in cardiomyocytes. - Gs activated a small subset of the Gq-induced genes, but was sensitive to the BET degrader molecule dBET6. - Distinct G protein-dependent effects may be conserved across cell types.

molecular biology↗

bioRxiv2024

The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization

Statement of SignificanceWe showed that loss of the orphan G protein-coupled receptor GPR52 in human breast cell lines leads to increased cell clustering, hybrid/partial EMT, and increased tumor burden in zebrafish. BackgroundG protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors that transmit critical signals from extracellular to intracellular spaces. Transcriptomic data of resected breast tumors show that low mRNA expression of orphan GPCR GPR52 correlates with reduced overall survival in patients with breast cancer, leading to the hypothesis that loss of GPR52 supports breast cancer progression. MethodsCRISPR-Cas9 was used to knockout GPR52 in the human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line MCF10A. 2D and 3D in vitro studies, electron microscopy, Matrigel culture, and a zebrafish xenograft model were used to assess the morphology and behavior of GPR52 KO cells. RNA-sequencing and proteomic analyses were also conducted on these cell lines, and transcriptomic data from The Cancer Genome Atlas (TCGA) database were used to compare GPR52-null and wild-type (WT) signatures in breast cancer. ResultsLoss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells, and zebrafish injected with GPR52 KO cells developed a greater total cancer area than those injected with control cells. RNA sequencing and proteomic analyses of GPR52-null breast cancer cells revealed an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates the production of cAMP, induces phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some GPR52 KO phenotypes. ConclusionGPR52 is an orphan GPCR and its role in cancer progression has not been previously characterized. We found that GPR52 loss in breast cancer cells can lead to increased cell clustering, collective invasion, and EMT in vitro. These are features of increased cancer aggression. Our results reveal that GPR52 loss is a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option for breast cancer. O_FIG O_LINKSMALLFIG WIDTH=169 HEIGHT=200 SRC="FIGDIR/small/604482v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@8b0f55org.highwire.dtl.DTLVardef@19bd139org.highwire.dtl.DTLVardef@c52117org.highwire.dtl.DTLVardef@1ffa2bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2023

Structure and dynamics of a pentameric KCTD5/Cullin3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by post-translational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain (CTD), engages CUL3 to form the central scaffold of a cullin- RING E3 ligase complex (CRL3KCTD5) that ubiquitylates G{beta}{gamma} and reduces G{beta}{gamma} protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/G{beta}1{gamma}2 assembly reveals a highly dynamic complex with rotations of over 60{degrees} between the KCTD5BTB/CUL3NTD and KCTD5CTD/G{beta}{gamma} moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate G{beta}{gamma} in an E3-E3 super-assembly, and extension of the structure to include full- length CUL3 with RBX1 and an ARIH1[~]ubiquitin conjugate reveals that some conformational states position the ARIH1[~]ubiquitin thioester bond to within 10 [A] of lysine-23 of G{beta} and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/G{beta}{gamma} complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex. Significance StatementIn humans, [~]600 enzyme complexes can carry out protein ubiquitylation, and the most abundant class of these are the cullin3-RING-ligase complexes (CRL3s). CRL3s are multiprotein complexes built around a BTB/cullin3 core, and the incorporation of different BTB proteins into this scaffold results in distinct architectures that ubiquitylate a wide range of substrates. In most cases, it is not known how the complexes are tuned to their substrates. We show that the BTB protein KCTD5 is the central organizer in a CRL3KCTD5 complex, and that the architecture and internal dynamics of KCTD5 are essential for positioning a G{beta}{gamma} substrate protein near an activated ubiquitin for the transfer reaction. This explains how KCTD5 targets G{beta}{gamma} for proteasomal degradation and regulates cellular activities.

biochemistry↗

bioRxiv2022

Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of {beta}-arrestin ({beta}arr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for {beta}arrs, agonist-stimulation first drives the translocation of {beta}arrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of {beta}arr translocation to endosomes although {beta}arrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like {beta}arr1 conformation, rescues endosomal translocation of {beta}arr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in {beta}arr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of {beta}arr1 with {beta}2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of {beta}arr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery. SignificanceThe interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and {beta}arrs have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of receptor-transducer complexes to encode gain of function has not been documented so far. Here, we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound {beta}arr1 can allosterically enhance endosomal trafficking of {beta}arr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like {beta}arr1 conformation and enhances the interaction of {beta}2-adaptin with {beta}arr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.

biochemistry↗

bioRxiv2021

Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.

physiology↗