Extracellular Ligand-binding Domain Research Articles

Structural basis of positive allosteric modulation of metabotropic glutamate receptor activation and internalization

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the functional and structural effects of orthosteric versus allosteric ligands. We find, using electrophysiological and live cell imaging assays, that both agonists and positive allosteric modulators (PAMs) can drive activation and internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling.

Molecular mechanism of ligand gating and opening of NMDA receptor.

Glutamate transmission and activation of ionotropic glutamate receptors are the fundamental means by which neurons control their excitability and neuroplasticity1. The N-methyl-D-aspartate receptor (NMDAR) is unique among all ligand-gated channels, requiring two ligands-glutamate and glycine-for activation. These receptors function as heterotetrameric ion channels, with the channel opening dependent on the simultaneous binding of glycine and glutamate to the extracellular ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, respectively2,3. The exact molecular mechanism for channel gating by the two ligands has been unclear, particularly without structures representing the open channel and apo states. Here we show that the channel gate opening requires tension in the linker connecting the LBD and transmembrane domain (TMD) and rotation of the extracellular domain relative to the TMD. Using electron cryomicroscopy, we captured the structure of theGluN1-GluN2B(GluN1-2B) NMDAR in its open state bound to a positive allosteric modulator. This process rotates and bends the pore-forming helices in GluN1 and GluN2B, altering the symmetry of the TMD channel from pseudofourfold to twofold. Structures of GluN1-2B NMDAR in apo and single-liganded states showed that binding of either glycine or glutamate alone leads to distinct GluN1-2B dimer arrangements but insufficient tension in the LBD-TMD linker for channel opening. This mechanistic framework identifies a key determinant for channel gating and a potential pharmacological strategy for modulating NMDAR activity.

The human-specific nicotinic receptor subunit CHRFAM7A reduces α7 receptor function in human induced pluripotent stem cells-derived and transgenic mouse neurons.

We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists.

Current and Emerging Treatment Options for HER2‑Positive Gastroesophageal Cancer

Gastroesophageal cancer (GEC) is the fifth most common cancer and the second most common cause of cancer-related mortality, with 1.3 million annual deaths worldwide. The global incidence is increasing, particularly among younger patients. GEC can be classified into subtypes based on anatomic location, histology, molecular characteristics, or tumour biology and genomics. In approximately 20% of all GECs overexpression of HER2 is identified. The landscape of treatment options in this patient population is evolving rapidly. This review summarizes the progress of HER2-directed therapies for advanced disease and highlights future directions in targeting the disease. The epidermal growth factor receptor (EGFR) family of transmembrane tyrosine kinase receptors, EGFR/HER1, HER2/neu, HER3, and HER4, all have an extracellular ligand-binding domain, lipophilic transmembrane domain, and an intracellular domain with tyrosine kinase activity, binding to these receptors results in activation of downstream RAS/MAPK and PI3K/AKT pathways. In turn, this induces cell proliferation, differentiation, migration, and survival. The phase III Trastuzumab for Gastric Cancer (ToGA) trial reported the incidence of HER2-positive gastric cancer to be 22%. Therefore, targeting HER2 and its downstream signaling pathways holds important potential as a therapeutic strategy. Figure 1 illustrates potential targeting mechanisms that will be discussed in this review.

Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-β 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of Gprotein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the Gprotein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and Gprotein binding.

Glu1022.53-Mediated Early Conformational Changes in the Process of Light-Induced Green Cone Pigment Activation.

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the β-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu1022.53 situated in TM2. The weakening of the hydrogen bond strength at Glu1022.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met862.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu1022.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Structural basis for excitatory neuropeptide signaling

Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily is diverse in terms of its gating stimuli, with some DEG/ENaCs gated by neuropeptides, and others gated by pH, mechanical force or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. Here we dissected the structural basis for neuropeptide-gated activity of a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. Structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes of channel gating, which we verified with complementary mutagenesis experiments. Our results illuminate channel gating in DEG/ENaCs and offer a structural template for experimental dissection of channel pharmacology and ion conduction.

Ex Vivo Drug Sensitivity Evaluation of a ZMYM2::FGFR1 Fusion-Positive 8p11 Myeloproliferative Syndrome (EMS) Leukemia

Introduction : Fibroblast growth factor receptors (FGFRs) are conserved single transmembrane receptor tyrosine kinases (RTKs) with an extracellular ligand-binding domain and a cytoplasmic tyrosine kinase domain. They play vital roles in cellular proliferation, survival, development, fetal organogenesis, metabolism, and tissue repair. 8p11 myeloproliferative syndrome (EMS) is a hematologic malignancy caused by a translocation of the fibroblast growth factor receptor 1 (FGFR1) gene on chromosome 8p11-12. The most common fusion partner of FGFR1 is the zinc-finger domain ZNF198 (also known as ZMYM2) on chromosome 13q12. Pemigatinib is approved for FGFR fusion positive myeloid/lymphoid neoplasms (MLNs), and other FGFR inhibitors are being tested in clinical trials. However, EMS patients still face a poor prognosis and drug resistance, necessitating further research to overcome these challenges. This study evaluated ex vivo drug screening assay data generated from ZMYM2::FGFR1 positive primary leukemia samples throughout the course of an EMS patient's clinical response and validated these findings on a ZMYM2::FGFR1 positive cell line. Methods : Ex vivo and drug screening experiments were performed on freshly isolated primary patient samples and a ZMYM2::FGFR1-expressing cell line. The fusion was confirmed using FISH, and additional clinical specimens were collected with informed patient consent. Ex vivo drug sensitivity assays were conducted on patient mononuclear cells isolated from blood and bone marrow. IL-3 dependent BaF3 cells were infected with retrovirus containing a ZMYM2::FGFR1-V5 construct. ZMYM2::FGFR1 transformed BaF3 cells were evaluated with single agent and drug combination panels. Sanger sequencing of the FGFR portion of the fusion was performed on patient samples to confirm the breakpoint and identify potential mutations affecting sensitivity to Ponatinib throughout the course of treatment. Results : Sample were obtained with informed consent from a 36-year-old man diagnosed with 8p11 myeloproliferative syndrome and a ZMYM2::FGFR1 fusion along with a RUNX1 R201Q mutation. Ex vivo drug screens performed on isolated mononuclear cells demonstrated sensitivity to Ponatinib and Pemigatinib. He underwent treatment with HyperCVAD plus Ponatinib and subsequently HyperCVAD with Ponatinib and Velcade (Bortezomib) to CR1, FISH for FGFR1 rearrangement was undetected. He was scheduled to proceed with hematopoietic stem cell transplant (HSCT), however his pre-transplant bone marrow biopsy showed return of his FGFR1 translocation by FISH. Ex vivo drug screening assays confirmed resistance to Ponatinib, but continued sensitivity to Pemigatinib. He was transitioned to treatment with single agent Pemigatinib and was able to re-clear his FGFR1 fusion by FISH, and has since undergone hematopoietic stem cell transplantation. He remains in remission on post-transplant Pemigatinib maintenance therapy. Sanger sequencing performed at the time of progression on Ponatinib identified a nucleotide alteration resulting in an amino acid substitution (FGFR1 F686L; ZMYM2::FGFR1 F1171L) within the kinase domain that could contribute to decreased Ponatinib sensitivity. Bortezomib and Axitinib exhibited high efficacy on the patient's sample. Ex vivo drug assays performed on ZMYM2::FGFR1 transformed BaF3 cells confirmed the efficacy of Pemigatinib and Ponatinib. Other FGFR inhibitors, including Olverematinib, AZD4547, Axitinib, Cediranib, Dovitinib, and Lenvatinib, also demonstrated exquisite sensitivity. Despite extensive screening, no other single agents or drug combinations exhibited increased effectiveness in the ZMYM2::FGFR1 transformed BaF3 cells except for Trametinib, a MEK inhibitor, and the combination of Belvarafenib (RAF inhibitor) and Gilteritinib (FLT3 inhibitor). Conclusions : Ex vivo drug sensitivity assays demonstrated the highly selective efficacy of FGFR inhibitors in ZMYM2::FGFR1 fusion-positive leukemia cells and a fusion-expressing BaF3 cell line. Mutations in the FGFR1 kinase domain (ZMYM2::FGFR1 F1171L) could contribute to Ponatinib insensitivity. These ex vivo drug screening results provide further support for ongoing clinical trials which are investigating the use of single agent Pemigatinib and other FGFR1 inhibitors for the treatment of patients with FGFR1-fusion positive leukemias.

Intracytoplasmatic BCMA Point Mutation and Teclistamab Resistance: Genomic Assessment of a Multidrug-Resistant Multiple Myeloma Patient

Introduction Proteasome inhibitors (PIs) are standard drugs for the treatment of Multiple Myeloma (MM), bispecific antibodies (BsAbs) and CART cells are rapidly and successfully being integrated into treatment regimens. Still, several current studies have shown that drug resistance remains one of the biggest clinical challenges. For instance, deletions in BCMA (alias TNFRSF17) were shown to confer a target loss and therefore resistance to BCMA CARTs (Da Via et al. Nat Med 2021, Samur et al. Nat Commun 2021) as well as to a CD3xBCMA bispecific T cell-engager (Truger et al. Blood Adv 2021). Besides the extracellular ligand-binding domain, BCMA further consist of a membrane spanning and a cytoplasmatic region that activates cell functions. To date not much is known in regard to alterations affecting the latter and their role in resistance. Here we present genetic profiling of a multi-refractory MM patient who relapsed to PIs, anti- BCMA (Teclistamab) and anti- GPRC5D (Talquetamab) BsAbs. Methods We performed FISH and a targeted capture-hybridization DNA sequencing panel (tchDNA-Seq) as previously described in Rosa-Rosa et al. Cancers 2022 in two sequential samples: first a bone marrow biopsy at diagnosis and second, 43 months later, a FFPE biopsy of the retroperitoneal mass of a progression plasmacytoma, after PI but prior Teclistamab treatment. The MM-specific tchDNA-Seq panel includes over 100 genes involved in MM progression, drug resistance, immunotherapy and canonical loci to cover IG rearrangements and frequent translocations. In order to predict the impact of the observed gene mutations on the protein structure, we generated 3D projections using the PyMOL molecular visualization system and references from the Protein Data Bank. The KinasePhos3 program was used to predict the most probable kinase to phosphorylate new, by the mutation generated, sites. Results Via FISH, the patient was stratified as a high-risk patient as 13% of the tumor cells showed a del17p at diagnosis, 97% a t(4;14) translocation, and 88% chromosomal gains for 1q21 and 11q13. The patient initially responded well to VRd+HCT. After 3.6 years he relapsed and developed an abdominal plasmacytoma. He was non-responsive to the following Isa-Kd treatment and relapsed again, two months after Teclistamab induction. To the subsequent PomCyDex and Talquetamab treatments he was primary refractory. In the plasmacytoma, four SNVs were detected: PSMC5 p.G152V with a VAF of 42%, BCMA p.C128Y (3%) and the two B2M mutations B2M p.S14F (47%) and B2M p.L107F (11%). At diagnosis, the VAF was 1% for PSMC5 p.G152V and B2M p.S14F, the other two SNVs were absent. In B2M, mutations in residues 13-16 have been associated with impaired protein expression and complex formation (Challa-Malladi et al. Cancer Cell 2011). The PSMC5 SNV was topologically identified within a glycine-rich loop in the ATP binding pocket of the proteasome in direct interaction with the ATP amine group, therefore likely impacting the proteolytic capacity increasing the PI tolerance. B CMA p.C128Y is located in the C-terminal cytoplasmatic tail within a highly conserved 25-aa protein segment (position 119 to 143) that has been shown to be necessary for TRAF association and NF-κB, Elk-1, and JNK activation (Hatzoglou et al. J Immunol 2000). A cysteine is mutated to a tyrosine. With high probability, this new site will be phosphorylated by kinases of the c-Src family (score 0.92), e.g. Fyn (0.71) or Lyn (0.66). Moreover, this new phosphotyrosine would become a binding site for the kinase SH2 domain (Diop et al. Int J Mol Sci 2022). Conclusions Here we report emerging mutations in PSMC5 and BCMA, that are likely causal for PI and Teclistamab resistance. PSMC5 p.G152 represents a mutational hotspot as four more PI-resistant patients with substitutions were recently reported in Haertle et al. Cancers 2023. The intracytoplasmatic BCMA SNV creates a new kinase phosphorylation and binding site resulting in aberrant downstream signaling. B2M forms part of the MHC1 molecules, responsible for antigen presentation to CD8+ T cells and the B2M serum level is a prognostic indicator in MM. However, the role of B2M alterations in disease progression and drug resistance needs to be elucidated. Predicting the biological impact based on 3D structural models remains speculative, thus, functional confirmation introducing the BCMA and B2M alterations in cell line models by genetic engineering is pending.

Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes.

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy.

Synthesis and Bioevaluation of 3-(Arylmethylene)indole Derivatives: Discovery of a Novel ALK Modulator with Antiglioblastoma Activities.

Glioblastoma is the most common brain tumor, with high recurrence and low survival rates. An integrative bioinformatics analysis demonstrated that anaplastic lymphoma kinase (ALK) is a promising therapeutic target for glioblastoma. We designed and synthesized a series of 3-(arylmethylene)indole derivatives, which were further evaluated for antiproliferative activity using glioma cell lines. Among them, compound 4a significantly inhibited the viability of glioblastoma cells. With favorable pharmacokinetic characteristics and blood-brain barrier permeability, 4a improved the survival rate and inhibited the growth of orthotopic glioblastoma. The Phospho-Totum system revealed that ALK was a potential target for the antiglioblastoma activity of 4a. Further experiments indicated that 4a might be a novel ALK modulator, which interacted with the extracellular ligand-binding domain of ALK, thus selectively induced ERK-mediated autophagy and apoptosis. Our findings provide an alternative ALK-based targeting strategy and a new drug candidate for glioblastoma therapy.

In-silico and structure-based assessment to evaluate pathogenicity of missense mutations associated with non-small cell lung cancer identified in the Eph-ephrin class of proteins.

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.

A novel EGFR variant EGFRx maintains glioblastoma stem cells through STAT5.

Glioblastomas are universally lethal brain tumors containing tumor-propagating glioblastoma stem cells (GSCs). EGFR gene amplification or mutation is frequently detected in GBMs and is associated with poor prognosis. However, EGFR variants in GSCs and their role in the maintenance of GSCs and progression of GBM are unclear. EGFR variants were detected through bioinformatic HISAT-StringTie-Ballgown pipeline and verified through 5' RACE, RT-PCR, ribonuclease protection, and northern blotting assays. EGFRx function was investigated through neurosphere, cell viability, intracranial xenograft and RNA-seq assays. EGFRx-STAT5 signaling was investigated through western blotting, coimmunoprecipitation, immunofluorescence, luciferase reporter, RT-PCR and CUT&Tag assays. We identified a novel EGFR variant (EGFRx), that is specifically expressed in GSCs. Unlike the EGFRvIII variant, which lacks exons 2-7, EGFRx is characterized by the absence of exons 2-14, and encodes an EGFR protein that does not possess the entire extracellular ligand-binding domain. We observed that EGFRx exhibits significant glycosylation, is required for GSC self-renewal, proliferation, and tumorigenesis, and highly active in glioblastomas compared to normal brain tissue. Mechanistically, EGFRx constitutively and specifically activates STAT5 in GSCs through spontaneous asymmetric dimerization of the kinase domain. EGFRx plays essential roles in the maintenance of the GSC phenotype through constitutive activation of STAT5 and promotes GBM progression, suggesting that EGFRx-STAT5 signaling represents a promising therapeutic target for GBM.

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Ionotropic glutamate receptors in GtoPdb v.2023.1

The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [36, 94, 157]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), 3 TM domains (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy- terminal domain (CTD) [101, 70, 109, 157, 84]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2- see below) has recently been solved at 3.6Å resolution [145] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [73]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [36, 68, 32, 79, 43, 116, 25, 67, 157, 114, 115, 165]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [29]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.NMDA receptorsNMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [42, 26]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [49, 101, 73]. However, more complex tri-heteromeric assemblies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca2+ and is blocked, in a voltage-dependent manner, by Mg2+ such that at resting potentials the response is substantially inhibited.AMPA and Kainate receptorsAMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [44, 105, 155, 66]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [89, 121, 120]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [121, 67, 120]). Kainate receptors may also exhibit 'metabotropic' functions [89, 133]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [120, 90]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl- for their activation [12, 122]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca2+; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [141, 65]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [89, 120]. Native AMPA and kainate receptors displaying differential channel conductances, Ca2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [31, 65, 93]. GluA1-4 can exist as two variants generated by alternative splicing (termed ‘flip’ and ‘flop’) that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [105]. Splice variants of GluK1-3 also exist which affects their trafficking [89, 120].

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors.

αD-conotoxins are 11kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein (Ls-AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls-AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

The SR protein RSP-2 influences expression of the truncated insulin receptor DAF-2B in Caenorhabditis elegans.

The alternatively spliced daf-2b transcript in Caenorhabditis elegans encodes a truncated isoform of the nematode insulin receptor that retains the extracellular ligand binding domain but lacks the intracellular signaling domain and is therefore unable to transduce a signal. To identify factors that influence expression of daf-2b, we performed a targeted RNA interference screen of rsp genes, which encode splicing factors from the serine/arginine protein family. Loss of rsp-2 significantly increased the expression of a fluorescent daf-2b splicing reporter, as well as increasing expression of endogenous daf-2b transcripts. Correspondingly, rsp-2 mutants exhibited similar phenotypes to those previously observed with DAF-2B overexpression, namely suppression of pheromone-induced dauer formation, enhancement of dauer entry in insulin signaling mutants, inhibition of dauer recovery, and increased lifespan. However, the epistatic relationship between rsp-2 and daf-2b varied according to the experimental context. Increased dauer entry and delayed dauer exit of rsp-2 mutants in an insulin signaling mutant background were partially dependent on daf-2b. Conversely, suppression of pheromone-induced dauer formation and increased lifespan in rsp-2 mutants were independent of daf-2b. These data demonstrate that C. elegans RSP-2, an ortholog of human splicing factor protein SRSF5/SRp40, is involved in regulating the expression of the truncated DAF-2B isoform. However, we also find that RSP-2 can influence dauer formation and lifespan independently of DAF-2B.