Biology subjects

Inoue, A.

Publications and source records attributed to Inoue, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

G protein-biased LPAR1 agonism promotes prototypic antidepressant effects

Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), in treating severe depression. However, the molecular mechanisms underlying the high efficacy of TCAs have not been completely understood. Herein, we found that lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, mediates the antidepressant effects of amitriptyline, a typical TCA. Amitriptyline directly bound to LPAR1 and activated downstream G protein signaling without affecting {beta}-arrestin signaling, which implied that amitriptyline could act as a G protein-biased agonist of LPAR1. This biased agonism is unique to TCAs and has not been observed in other antidepressants, such as SSRIs. Long-term infusion of mouse hippocampus with 1-oleoyl-2-O-methyl-glycerophosphothionate (OMPT), a potent G protein-biased LPAR1 agonist, induced behavior similar to that induced by antidepressants. In contrast, LPA, a non-biased agonist of LPAR1, induced anxious behavior, indicating that LPAR1 may regulate conflicting emotional behaviors because of the downstream signaling bias. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in hippocampus. Ingenuity pathway analysis indicated that chronic intrahippocampal administration of OMPT could activate LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. The results reveal the unique antidepressant effects of TCAs and indicate the potential of G protein-biased agonists of LPAR1 as targets for novel antidepressants.

neuroscience↗

bioRxiv2022

Endosomal Chemokine Receptor Signalosomes Regulate Central Mechanisms Underlying Cell Migration

Chemokine receptors are GPCRs that regulate chemotactic migration of a wide variety of cells including immune and cancer cells. Most chemokine receptors contain features associated with the ability to stimulate G protein signaling during {beta}-arrestin-mediated receptor internalization into endosomes. As endosomal signaling of certain non-GPCR receptors plays a major role in cell migration, we chose to investigate the potential role of endosomal chemokine receptor signaling on mechanisms governing this function. Applying a combination of pharmacological and cell biological approaches, we demonstrate that the model chemokine receptor CCR7 recruits G protein and {beta}-arrestin simultaneously upon chemokine stimulation, which enables internalized receptors to activate G protein from endosomes. Furthermore, spatiotemporal-resolved APEX2 proteome profiling shows that endosomal CCR7 uniquely enriches specific Rho GTPase regulators as compared to plasma membrane CCR7, which is directly associated with enhanced activity of the Rho GTPase Rac1 and chemotaxis of immune T cells. As Rac1 drives the formation of membrane protrusions during chemotaxis, our findings suggest an important integrated function of endosomal chemokine receptor signaling in cell migration.

cell biology↗

bioRxiv2022

GPCRome-wide structural analysis of G-protein-coupling selectivity

We present a comprehensive computational analysis of available 3D GPCR-G-protein complexes to inspect the structural determinants of G-protein-coupling selectivity. Analysis of the residue contacts at interaction interfaces has revealed a network of secondary structure elements recapitulating known structural hallmarks determining G-protein-coupling specificity, including TM5, TM6 and ICLs. We coded interface contacts into generic-number fingerprints to reveal specific coupling-determinant positions. Clustering of Gs vs Gi complexes is best achieved when considering both GPCR and G-protein contacting residues rather than separated representations of the interaction partners, suggesting that coupling specificity emerges as contextual residue interactions at the interface. Interestingly, Gs-GPCR complexes contain a higher number of contacts than Gi/o-GPCR complexes, likely caused by overall higher conservation and structural constraint on the Gs interface. In contrast, Gi/o proteins adopt a wider number of alternative docking poses on cognate receptors, as assessed via structural alignments of representative 3D complexes. Furthermore, binding energy calculations demonstrate that distinct structural properties of the complexes contribute to higher stability of Gs than Gi/o complexes. AlphaFold2 predictions of experimental binary complexes confirmed several of these structural features and allowed us to augment the structural coverage of poorly characterized complexes (e.g. G12/13). We propose that the structural properties of different G-protein complexes, such as structural restraining of Gs compared to Gi/o ones, could be instrumental in fine-tuning their activation and downstream signaling mechanisms. Highlights-Comprehensive structural bioinformatics analysis of available GPCR-G-protein complexes captures common as well as group-specific structural features responsible of receptor-G-protein recognition -Distinct contact patterns explain different docking modes of Gi/o vs Gs complexes, the latter being characterized by higher enrichment of characteristic contacts and lower structural variability suggestive of higher interface conservation. -Structural hallmarks are associated with different estimated binding energies, which mainly discriminates Gs versus Gi/o couplings, but which also point to class-dependent differences (e.g. Class A vs Class B) in binding the same transducer (Gs)

bioinformatics↗

bioRxiv2022

Ligands tune the local and global motions of neurotensin receptor 1 (NTS1): a DFT-guided solution NMR analysis

Unlike many signaling proteins that function as binary switches between on and off states, G protein-coupled receptors (GPCRs) exhibit basal activity that can be increased or decreased by numerous ligands. A given receptor can recognize multiple ligands, allosteric modulators, and transducers to create a complex free energy landscape. Many of the lowest energy states have been captured by static structural techniques while detailing the wells widths, metastable states, and the transition between them, is still in its infancy. Nuclear magnetic resonance (NMR) spectroscopy can monitor the structure and dynamics of GPCR ensembles across fifteen orders-of-magnitude, but technical challenges have limited its application to super-microsecond timescales. Focusing on a prototypical peptide-binding GPCR, the neurotensin receptor 1 (NTS1), we employed NMR and density functional theory (DFT) to probe global sub-nanosecond motions. The near random coil chemical shifts of the apo receptor produced a poor correlation with theoretical predictions that may indicate a high degree of conformational averaging in solution, a crystallization artifact, or both. Whereas orthosteric agonists and antagonists both rigidified the receptor, but to varying degrees, which suggests conformational entropy differentially contributes to their respective pharmacology. The strong correlations of observed and theoretical chemical shifts lend confidence to interpreting spectra in terms of local structure, methyl dihedral angle geometry, and pico-second timescale transitions. Together, our results suggest a role for sub-nanosecond dynamics and conformational entropy in GPCR ligand discrimination.

biophysics↗

bioRxiv2022

Activation of the urotensin-II receptor by anti-COVID-19 drug remdesivir induces cardiomyocyte dysfunction

Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular (CV) side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor (GPCR) screening in combination with structural modeling and found that remdesivir is a selective agonist for urotensin-II receptor (UTS2R). Functionally, remdesivir treatment induced prolonged field potential in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and reduced contractility in neonatal rat cardiomyocytes, both of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants (SNVs) in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related CV events and that genetic variations of UTS2R gene can be a potential risk factor for CV events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future. One Sentence SummaryRemdesivir s activity as a selective agonist of urotensin-II receptor underlies its known cardiotoxicity in anti-viral therapy.

pharmacology and toxicology↗

bioRxiv2022

Modulation of cannabinoid receptor signaling by endocannabinoids

Endocannabinoids (eCBs) are endogenous lipid molecules that activate the cannabinoid receptor 1 (CB1), a G protein coupled receptor (GPCR) that signals primarily through the Gi/o family of G proteins to regulate neurotransmitter release. Consequently, CB1 is an important therapeutic target for several neurological disorders. How eCBs interact with CB1 is not known and the downstream signaling they activate is not well understood. In this study we show that eCBs do not activate Gi1 as much as synthetic cannabinoids. To characterize activation of CB1 by eCB, we formed an eCB analogue-bound (AMG315) CB1-Gi signaling complex for structural studies. The structure reveals differences in the orthosteric ligand binding pocket not seen in the previous CB1 structures, providing insights into the structural determinants of ligand efficacy. In combination with signaling and simulation data, this study provides mechanistic insights into CB1 activation by different classes of ligands, and sheds light on the G protein preferences between endogenous and exogenous ligands.

biochemistry↗

bioRxiv2022

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore novel aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, which share a common chemokine agonist (CXCL12), in terms of their G-protein coupling, {beta}-arrestin ({beta}arr) recruitment, contribution of GRKs, and ERK1/2 MAP kinase activation. We observe that CXCR7 lacks G-protein coupling while maintaining robust {beta}arr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected, however, it exhibits significantly reduced {beta}arr-coupling compared to CXCR7 in response to their shared natural agonist, CXCL12. These two receptors induce distinct {beta}arr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We further determine the crystal structure of {beta}arr2 in complex with a carboxyl-terminal phosphopeptide derived from CXCR7, which reveals a smaller interdomain rotation than observed previously for activated {beta}arrs. Importantly, structure-guided cellular experiments reveal a key contribution of a single phosphorylation site in CXCR7 on {beta}arr recruitment and endosomal trafficking. Taken together, our study provides molecular insights into intrinsic bias encoded in the CXCR4-CXCR7 system, and it has broad implications for therapeutically important framework of biased agonism.

biochemistry↗

bioRxiv2022

Conservation and divergence of canonical and non-canonical imprinting in murids

BackgroundGenomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behaviour. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we used low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extra-embryonic tissues at allele-specific resolution. ResultsWe identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse revealed that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extra-embryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extra-embryonic imprinting. Meta-analysis of novel imprinted genes revealed multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the birth of ZFP57 binding motifs and the insertion of endogenous retroviral promoters. ConclusionsIn summary, we provide a comprehensive list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

genomics↗

bioRxiv2022

Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

High-density lipoprotein (HDL) particles suppress inflammation-induced tissue injury via vascular and myeloid cell-dependent mechanisms. As such, HDL-associated bioactive lipids such as sphingosine 1-phosphate (S1P) and prostacyclin (PGI2) signal via their respective G protein-coupled receptors on target cells to promote vascular endothelial function and suppress platelet and myeloid-dependent pathophysiology. Here we have constructed a fusion protein of apolipoprotein A1 (ApoA1) and apolipoprotein M (ApoM) (A1M) that forms HDL-like particles and chaperones S1P and Iloprost, stable PGI2 analog. The A1M/S1P complex activates S1P receptor-1 (S1PR1) as a Gi-biased agonist and attenuates the inflammation-induced NF{kappa}B pathway while A1M/Iloprost acts via IP receptor to inhibit platelet aggregation and promote endothelial barrier function. In addition to enhancing the endothelial barrier, A1M/S1P suppresses neutrophil influx, oxidative burst and inflammatory mediator secretion in a sterile inflammation model. We propose that A1M could be useful as a therapeutic to induce S1P and PGI2-dependent anti-inflammatory functions and suppress collateral tissue injury.

biochemistry↗

bioRxiv2021

The Effect of Ligands and Transducers on the Neurotensin Recep-tor 1 (NTS1) Conformational Ensemble

Using a discrete, intracellular 19F-NMR probe on transmembrane helix 6 (TM6) of the Neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptors structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the ms-s timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other GPCRs, the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to {beta}-arrestin-1 or the C-terminal helix of Gq, which comprises 60% of the GPCR/G protein interface surface area. Whereas {beta}-arrestin-1 exclusively selects for pre-existing active-like substates, the Gq peptide induces a new substate. Both transducer molecules promote substantial line-broadening of active-like states suggesting contributions from additional s-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and ii) the available static structures do not represent the entire conformational ensemble observed in solution.

biophysics↗

bioRxiv2021

Structural insights into distinct signaling profiles of the μOR activated by diverse agonists

Drugs targeting the G protein-coupled -opioid receptor (OR) are the most effective analgesics available but are also associated with fatal respiratory depression. While some partial opioid agonists appear to be safer than full agonists, the signaling pathways responsible for respiratory depression have yet to be elucidated. Here we investigated the structural and mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two OR agonists with different safety profiles. LFT, one of the most potent and lethal opioids, and MP, a derivative from the kratom plant with reduced respiratory depression in animal studies at equianalgesic doses, exhibited markedly different signaling efficacy profiles for G protein subtype activation and recruitment of {beta}-arrestins. Cryo-EM structures of the OR-Gi1 complex with MP (2.5[A]) and LFT (3.2[A]) revealed that the two ligands engage distinct sub-pockets, and molecular dynamics (MD) simulations showed additional differences in the binding site that propagate to the intracellular side of the receptor where G proteins and {beta}-arrestins bind. While MP favors the precise G protein-bound active state observed in the cryo-EM structures, LFT favors a distinct active state. These results highlight how drugs engaging different parts of the OR orthosteric pocket can lead to distinct signaling outcomes.

biophysics↗

bioRxiv2021

Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

Mini-G proteins are engineered thermostable variants of G subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in {beta}-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of G subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.

cell biology↗

bioRxiv2021

Membrane phosphoinositides stabilize GPCR-arrestin complexes and offer temporal control of complex assembly and dynamics

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized some GPCRs may complex with arrestin, while others interact transiently; this difference affects receptor signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one requiring PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a receptor core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.

biochemistry↗

bioRxiv2021

Integrative analysis of scRNAs-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator

Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by transcriptional regulator AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected by using Fucci technology express a minimal level of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit amplifying Aire+mTEC precursors during Aire+mTEC differentiation process of the postnatal thymus.

immunology↗

bioRxiv2021

GPCR-G protein selectivity - a unified meta-analysis

Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein couplome. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs.

pharmacology and toxicology↗

bioRxiv2021

Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

ObjectivesReceptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon is important for carbohydrate and lipid metabolism. MethodsSubcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking assays and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking Wiskott Aldrich Syndrome protein and scar homologue (WASH) complex were used to investigate the effect of a halt in recycling of internalised proteins on GCGR signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 up-regulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. ResultsGCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular retention of glucagon-stimulated GCGR in cells lacking WASH complex indicates that activated GCGRs undergo continuous cycles of internalisation and recycling despite apparent GCGR plasma membrane localisation up to 40 minutes post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist-stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from {beta}-arrestin-2 recruitment coupled to increased activation of Gs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only minor disturbance of carbohydrate metabolism was observed in mice with up-regulated hepatic RAMP2. ConclusionsBy retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted. Graphical abstractIcons sourced from [1] O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2021

A genetic mouse model with postnatal Nf1 and p53 loss recapitulates the histology and transcriptome of human malignant peripheral nerve sheath tumor

BackgroundMalignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas. Somatic inactivation of NF1 and cooperating tumor suppressors, including CDKN2A/B, PRC2, and p53, is found in most MPNST. Inactivation of the LATS1/2 kinases of the Hippo pathway was recently shown to cause tumors resembling MPNST histologically, although Hippo pathway mutations are rarely found in MPNST. Because existing genetically engineered mouse (GEM) models of MPNST do not recapitulate some of the key genetic features of human MPNST, we aimed to establish a mouse MPNST model that recapitulated the human disease genetically, histologically, and molecularly. MethodsWe combined two genetically modified alleles, an Nf1;Trp53 cis-conditional allele and an inducible Plp-CreER allele (NP-Plp), to model the somatic, possibly postnatal, mutational events in human MPNST. We also generated conditional Lats1;Lats2 knockout mice. We performed histopathologic analysis of mouse MPNST models and transcriptomic comparison of mouse models and human nerve sheath tumors. ResultsPostnatal Nf1;Trp53 cis-deletion resulted in GEM-MPNST that was histologically more similar to human MPNST than the widely used germline Nf1;Trp53 cis-heterozygous (NPcis) model and showed partial loss of H3K27me3. At the transcriptome level, Nf1;p53-driven GEM-MPNST were distinct from Lats-driven GEM-MPNST and resembled human MPNST more closely than do Lats-driven tumors. ConclusionsThe NP-Plp model recapitulates human MPNST genetically, histologically, and molecularly. Key PointsO_LIPostnatal Nf1;p53 cis-deletion in NP-Plp mice results in tumors similar to MPNST. C_LIO_LIThe transcriptomes of Nf1;p53-driven and Lats-driven MPNST models are distinct. C_LIO_LINP-Plp model resembles human MPNST genetically, histologically, and molecularly. C_LI Importance of the StudyMalignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with a poor prognosis and limited treatment options. Existing genetically engineered mouse (GEM) models of MPNST do not recapitulate some of the key genetic features of human MPNST. To model the somatic, possibly postnatal, mutational events seen in MPNST patients, we generated a GEM-MPNST model by combining two genetically modified alleles, an Nf1;Trp53 cis-conditional allele and a Plp-CreER allele. Our histologic and transcriptomic analyses showed that this NP-Plp model resembles human MPNST genetically, histologically, and molecularly--more so than the widely used NPcis model and the recently published Lats-driven model. The NP-Plp model is genetically simple, making it easy to maintain and an ideal platform for preclinical studies. Given its tamoxifen-inducible nature, this model can be used to study the time/stage dependency of the tumorigenic potential of Schwann cells.

cancer biology↗

bioRxiv2021

RAMPs regulate signalling bias and internalisation of the GIPR

Gastric inhibitory polypeptide (GIP) receptor is a class B1 GPCR, that responds to GIP and physiologically potentiates glucose-stimulated insulin secretion. Like most class B1 GPCRs, GIPR has been shown to interact with RAMPs, yet the effects of RAMPs on its signalling and trafficking remain poorly understood. We demonstrate that RAMPs modulate G protein activation and GIPR internalisation profiles. RAMP3 reduced GIPR Gs activation and cAMP production but retained GIPR at the cell surface, and this was associated with prolonged ERK1/2 phosphorylation and {beta}-arrestin association. By contrast, RAMP1/2 reduced Gq/11/15 activation of the GIPR. Through knockout mice studies, we show that RAMP1 is important to the normal physiological functioning of GIPR to regulate blood glucose levels. Thus, RAMPs act on G protein/{beta}-arrestin complexes, having both acute and chronic effects on GIPR function, while this study also raises the possibility of a more general role of RAMP3 to enhance GPCR plasma membrane localisation.

pharmacology and toxicology↗