receptor internalization
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2022 ◽  
Author(s):  
Dylan Scott Eiger ◽  
Noelia Boldizsar ◽  
Christopher Cole Honeycutt ◽  
Julia Gardner ◽  
Stephen Kirchner ◽  
...  

Some G protein-coupled receptor (GPCR) ligands act as biased agonists which preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the chemokine GPCR CXCR3. The signaling profile of CXCR3 changed as it trafficked from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling was critical for biased activation of G proteins, β-arrestins, and ERK1/2. In CD8+ T cells, the chemokines promoted unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation was dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2084
Author(s):  
Akito Endo ◽  
Shoichiro Asayama

The lactosylated poly(1-vinylimidazole) (PVIm-Lac) with various lactosylated degrees has been synthesized for the co-delivery of zinc ions (Zn) and plasmid DNA (pDNA). The Zn/DNA/PVIm-Lac complex formation has achieved the specific delivery of zinc ions to HepG2 cells. Especially, the resulting hepatocyte-specific delivery of zinc ions has increased the number of insulin receptors on the cell surface. Consequently, the Zn/DNA/PVIm-Lac complexes have suppressed insulin receptor internalization on the surface of the HepG2 cells, expecting to offer unique therapy to inhibit hepatic insulin clearance.


iScience ◽  
2021 ◽  
pp. 103539
Author(s):  
Julia Cappelli ◽  
Pamela Khacho ◽  
Boyang Wang ◽  
Alexandra Sokolovski ◽  
Wafae Bakkar ◽  
...  

Author(s):  
Luke S. Watson ◽  
Brynna Wilken-Resman ◽  
Alexus Williams ◽  
Guadalupe Sanchez ◽  
Taylor Lowry McLeod ◽  
...  

Insulin receptors are internalized by endothelial cells; however, the impact of hyperinsulinemia on this process is not known. Thus, the aim of this study is to determine the role of hyperinsulinemia on insulin receptor function and internalization, as well as the potential impact of protein tyrosine phosphatase 1B (PTP1B). To this end, hippocampal microvessels were isolated from male C57Bl/6J mice on either a control or high-fat diet and assessed for insulin receptor signaling. Cell surface insulin receptors in brain microvascular endothelial cells were labelled with biotin to assess the role hyperinsulinemia plays on receptor internalization in response to stimulation, with and without Claramine treatment, a potent PTP1B antagonist. Our results indicated that insulin receptor levels increased in tandem with insulin receptor dysfunction in the high-fat diet mouse hippocampal microvessels. Hyperinsulinemic cell-receptors demonstrate a shift in splice variation towards decreased IR-A/IR-B ratios and demonstrate a higher membrane-localized proportion. This corresponded with decreased autophosphorylation at sites critical for receptor internalization and signaling, however, Claramine restored signaling and receptor internalization in hyperinsulinemic cells. In conclusion, hyperinsulinemia negatively impacts brain microvascular endothelial cell insulin receptor function and internalization, likely through both alternative splicing and increased negative feedback from PTP1B.


Author(s):  
Virginia M. Smith ◽  
Huan Nguyen ◽  
John W. Rumsey ◽  
Christopher J. Long ◽  
Michael L. Shuler ◽  
...  

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01–20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2063-2063
Author(s):  
Katarina Zmajkovicova ◽  
Sabine Maier-Munsa ◽  
Barbara Maierhofer ◽  
Arthur G. Taveras ◽  
Adriana Badarau

Abstract Background: WHIM (Wart, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency with neutropenia and lymphopenia. The clinical presentation may include recurrent infections, and increased susceptibility to human papillomavirus. In >80% of cases, WHIM syndrome is caused by heterozygous gain-of-function (GOF) mutations in C-X-C chemokine receptor 4 (CXCR4), with >12 variants reported in WHIM syndrome to date (nonsense [NS], frameshift [FS], and missense[MS]) spanning 27 C-terminal amino acids . These mutations cause hyperactivation of downstream signaling and retention of WBC in the bone marrow (McDermott D, et al. Immunol Rev. 2019;287(1):91-102; Beaussant S, et al. Orphanet J Rare Dis. 2012;7(71):1-14). To date, a comprehensive study characterizing the functional abnormalities caused by pathogenic CXCR4 mutations and correlating these measures with clinical presentation in patients has not been conducted. Here, we aimed to establish genotype-phenotype correlations for all known pathogenic variants using in vitro functional assays. These assays characterize CXCR4 receptor trafficking and downstream signaling, which will enable the long-term goal of assessing pathogenicity of novel CXCR4 variants of uncertain significance (VUS). We further aimed to assess the in vitro response of each variant to mavorixafor, an investigational CXCR4 antagonist. Methods: We used the CXCR4-negative K562 cell line as a model system to express all 14 known CXCR4 variants identified in patients diagnosed with WHIM syndrome (previous reports, ClinVar, and genetic screening initiatives [Invitae PATH4WARD]). The effects of the mutations on CXCR4 receptor trafficking (internalization and degradation), downstream signaling (Ca 2+ mobilization, cAMP inhibition, ERK and AKT activation), and chemotaxis were studied in parallel in a series of assays in cells stimulated with the natural ligand CXCL12. All in vitro functional parameters characterized were investigated for potential correlations with the clinical phenotypes reported for each variant, including disease manifestations and biomarkers. Results: The most conserved feature of the 14 CXCR4 variants was an impaired receptor internalization in response to CXCL12, evidenced by higher percentage of CXCR4 receptors remaining on the cell surface compared to untreated control, with truncated variants showing maximum impairment and the MS variant E343K being least affected. The decreased CXCR4 internalization correlated with both decreased CXCR4 degradation and increased cAMP inhibition. When stimulated with CXCL12, most variants demonstrated a higher amplitude and duration of ERK and AKT activation. Chemotactic responses to CXCL12 were diverse, depending on the variant sequence and subtype. While Ca 2+ mobilization was not enhanced compared to wild-type (WT) CXCR4-expressing cells in this assay, mavorixafor demonstrated inhibition of Ca 2+ mobilization in all CXCR4 mutant cells with a trend toward greater effect in the NS variants. In addition, both ERK and AKT activation decreased with increasing concentration of mavorixafor. Correlation analyses of the functional parameters in cells expressing mutated or WT CXCR4 and clinical manifestations or WBC counts in patients with these mutations revealed that the CXCR4 internalization defect strongly correlated with severity of peripheral blood cytopenias (ie, decreases in absolute neutrophil counts, and in CD3+ and CD4+ T-cell levels), which was paralleled by an increased susceptibility to recurrent infections. In addition, AKT hyperactivation correlated with lower IgA and decreased CD4+ T-cell levels (Figure). Conclusions: In the current study, we performed a detailed functional analysis of the entire spectrum of CXCR4 WHIM mutations known to date. In vitro CXCR4 receptor internalization correlates with WBC cytopenias and an increased susceptibility to recurrent infections in patients with CXCR4 GOF mutations. These data suggest that CXCR4 internalization and AKT activation may be used as key assays for the assessment of CXCR4 variant pathogenicity in vitro and potentially as WHIM-related disease biomarkers. Additionally, all tested CXCR4 variant cell lines were sensitive to mavorixafor at clinically relevant concentrations, rescuing defective GOF signaling toward that of WT CXCR4-expressing cells. Figure 1 Figure 1. Disclosures Zmajkovicova: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Maier-Munsa: X4 Pharmaceuticals: Current Employment. Maierhofer: X4 Pharmaceuticals: Current Employment. Taveras: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Badarau: X4 Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months.


2021 ◽  
Author(s):  
Hiromi Watari ◽  
Hiromu Kageyama ◽  
Nami Masubuchi ◽  
Hiroya Nakajima ◽  
Kako Onodera ◽  
...  

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism remains unstudied because no external agonist has been discovered. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC). ThC-induced activation persists due to limited receptor internalization. Strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. We show that MPL is subject to sugar-mediated activation and that lectin-mediated activation kinetics differ from cytokine-mediated activation kinetics. Our data demonstrated the potential of lectins to provide deeper insight into human pathogenesis.


2021 ◽  
Vol 12 ◽  
Author(s):  
Duncan Kirby ◽  
Baljyot Parmar ◽  
Sepehr Fathi ◽  
Sagar Marwah ◽  
Chitra R. Nayak ◽  
...  

The Type I Interferon family of cytokines all act through the same cell surface receptor and induce phosphorylation of the same subset of response regulators of the STAT family. Despite their shared receptor, different Type I Interferons have different functions during immune response to infection. In particular, they differ in the potency of their induced anti-viral and anti-proliferative responses in target cells. It remains not fully understood how these functional differences can arise in a ligand-specific manner both at the level of STAT phosphorylation and the downstream function. We use a minimal computational model of Type I Interferon signaling, focusing on Interferon-α and Interferon-β. We validate the model with quantitative experimental data to identify the key determinants of specificity and functional plasticity in Type I Interferon signaling. We investigate different mechanisms of signal discrimination, and how multiple system components such as binding affinity, receptor expression levels and their variability, receptor internalization, short-term negative feedback by SOCS1 protein, and differential receptor expression play together to ensure ligand specificity on the level of STAT phosphorylation. Based on these results, we propose phenomenological functional mappings from STAT activation to downstream anti-viral and anti-proliferative activity to investigate differential signal processing steps downstream of STAT phosphorylation. We find that the negative feedback by the protein USP18, which enhances differences in signaling between Interferons via ligand-dependent refractoriness, can give rise to functional plasticity in Interferon-α and Interferon-β signaling, and explore other factors that control functional plasticity. Beyond Type I Interferon signaling, our results have a broad applicability to questions of signaling specificity and functional plasticity in signaling systems with multiple ligands acting through a bottleneck of a small number of shared receptors.


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