Cell Surface Levels Of Receptors Research Articles

Multiple mechanisms contribute to acquired TRAIL resistance in multiple myeloma

Multiple Myeloma (MM) prognosis has recently improved thanks to the incorporation of new therapies to the clinic. Nonetheless, it is still a non-curable malignancy. Targeting cancer cells with agents inducing cell death has been an appealing alternative investigated over the years, as is the case of TRAIL, an agonist of DR4 and DR5 death receptors. This pathway, involved in apoptosis triggering, has demonstrated efficacy on MM cells. In this research, we have investigated the sensitivity of a panel of MM cells to this agent and generated TRAIL-resistant models by continuous culture of sensitive cells with this peptide. Using genomic and biochemical approaches, the mechanisms underlying resistance were investigated. In TRAIL-resistant cells, a strong reduction in cell-surface receptor levels was detected and impaired the apoptotic machinery to respond to the treatment, enabling cells to efficiently form the Death Inducing Signalling Complex. In addition, an upregulation of the inhibitory protein c-FLIP was detected. Even though the manipulation of these proteins was able to modify cellular responses to TRAIL, it was not complete, pointing to other mechanisms involved in TRAIL resistance.

Insights into Immune Exhaustion in Chronic Hepatitis B: A Review of Checkpoint Receptor Expression.

Hepatitis B, caused by the hepatitis B virus (HBV), often progresses to chronic infection, leading to severe complications, such as cirrhosis, liver failure, and hepatocellular carcinoma. Chronic HBV infection is characterized by a complex interplay between the virus and the host immune system, resulting in immune cell exhaustion, a phenomenon commonly observed in chronic viral infections and cancer. This state of exhaustion involves elevated levels of inhibitory molecules, cells, and cell surface receptors, as opposed to stimulatory counterparts. This review aims to elucidate the expression patterns of various co-inhibitory and co-stimulatory receptors on immune cells isolated from chronic hepatitis B (CHB) patients. By analyzing existing data, the review conducts comparisons between CHB patients and healthy adults, explores the differences between HBV-specific and total T cells in CHB patients, and examines variations between intrahepatic and peripheral immune cells in CHB patients. Understanding the mechanisms underlying immune exhaustion in CHB is crucial for developing novel immunotherapeutic approaches. This detailed analysis sheds light on the immune exhaustion observed in CHB and lays the groundwork for future combined immunotherapy strategies aimed at leveraging checkpoint receptors to restore immune function and improve clinical outcomes.

Visual feedback neurons fine-tune Drosophila male courtship via GABA-mediated inhibition

Many species of animals use vision to regulate their social behaviors. However, the molecular and circuit mechanisms underlying visually guided social interactions remain largely unknown. Here, we show that the Drosophila ortholog of the human GABAA-receptor-associated protein (GABARAP) is required in a class of visual feedback neurons, lamina tangential (Lat) cells, to fine-tune male courtship. GABARAP is a ubiquitin-like protein that maintains cell-surface levels of GABAA receptors. We demonstrate that knocking down GABARAP or GABAAreceptors in Lat neurons or hyperactivating them induces male courtship toward other males. Inhibiting Lat neurons, on the other hand, delays copulation by impairing the ability of males to follow females. Remarkably, the fly GABARAP protein and its human ortholog share a strong sequence identity, and the fly GABARAP function in Lat neurons can be rescued by its human ortholog. Using invivo two-photon imaging and optogenetics, we reveal that Lat neurons are functionally connected to neural circuits that mediate visually guided courtship pursuits in males. Our work identifies a novel physiological function for GABARAP in regulating visually guided courtship pursuits in Drosophila males. Reduced GABAA signaling has been linked to social deficits observed in the autism spectrum and bipolar disorders. The functional similarity between the human and the fly GABARAP raises the possibility of a conserved role for this gene in regulating social behaviors across insects and mammals.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Myeloid cell-targeted therapies for solid tumours.

Myeloid cells are the most abundant immune components of the tumour microenvironment, where they have a variety of functions, ranging from immunosuppressive to immunostimulatory roles. The myeloid cell compartment comprises many different cell types, including monocytes, macrophages, dendritic cells and granulocytes, that are highly plastic and can differentiate into diverse phenotypes depending on cues received from their microenvironment. In the past few decades, we have gained a better appreciation of the complexity of myeloid cell subsets and how they are involved in tumour progression and resistance to cancer therapies, including immunotherapy. In this Review, we highlight key features of monocyte and macrophage biology that are being explored as potential targets for cancer therapies and what aspects of myeloid cells need a deeper understanding to identify rational combinatorial strategies to improve clinical outcomes of patients with cancer. We discuss therapies that aim to modulate the functional activities of myeloid cell populations, impacting their recruitment, survival and activity in the tumour microenvironment, acting at the level of cell surface receptors, signalling pathways, epigenetic machinery and metabolic regulators. We also describe advances in the development of genetically engineered myeloid cells for cancer therapy.

Implication of thyroid hormone receptors in methamphetamine neurocognitive effects

Methamphetamine (MA) induces neurocognitive effects via several mechanisms. In the present study, we investigated the alteration of thyroid hormone receptor’s expression in the context of MA-induced memory impairment and explored the protective effects of exogenous thyroid hormones (THs). Male wistar rats, received increasing regimen of MA (1–10 mg/kg, intraperitoneal, twice a day for 10 days), were treated with T3 (40 μg/rat/day; intranasal, 2.5 μl/nostril) or T4 (20 µg/kg/day; intraperitoneal) for 7 days after MA cessation. All rats were subjected to novel object recognition memory test and then the mRNA levels of TH nuclear receptors (TRα1 and TRβ1) and seladin-1, an anti-apoptotic factor, and the protein level of TH cell surface receptor (integrin αvβ3) were measured in the hippocampus of rats. Our results showed that MA-induced memory impairment is concomitant with decreased level of TRα1 mRNA. T3 or T4 treatment significantly alleviated MA-induced memory impairment, but had no significant effect on the mRNA levels of TH nuclear receptors. However, T4 treatment significantly increased the protein level of cell surface receptor (αv subunit) in MA-treated rats. These findings suggest that MA neurocognitive effects can be associated with impaired TH signaling in the brain and introduce this pathway as a promising therapeutic approach against MA-induced memory impairment.

Liver macrophages and inflammation in physiology and physiopathology of non-alcoholic fatty liver disease.

Non‐alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, being a common comorbidity of type 2 diabetes and with important links to inflammation and insulin resistance. NAFLD represents a spectrum of liver conditions ranging from steatosis in the form of ectopic lipid storage, to inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Macrophages that populate the liver play important roles in maintaining liver homeostasis under normal physiology and in promoting inflammation and mediating fibrosis in the progression of NAFLD toward to NASH. Liver macrophages are a heterogenous group of innate immune cells, originating from the yolk sac or from circulating monocytes, that are required to maintain immune tolerance while being exposed portal and pancreatic blood flow rich in nutrients and hormones. Yet, liver macrophages retain a limited capacity to raise the alarm in response to danger signals. We now know that macrophages in the liver play both inflammatory and noninflammatory roles throughout the progression of NAFLD. Macrophage responses are mediated first at the level of cell surface receptors that integrate environmental stimuli, signals are transduced through multiple levels of regulation in the cell, and specific transcriptional programmes dictate effector functions. These effector functions play paramount roles in determining the course of disease in NAFLD and even more so in the progression towards NASH. The current review covers recent reports in the physiological and pathophysiological roles of liver macrophages in NAFLD. We emphasise the responses of liver macrophages to insulin resistance and the transcriptional machinery that dictates liver macrophage function.

Zebrafish Scube1 and Scube2 cooperate in promoting Vegfa signalling during embryonic vascularization.

The secreted and membrane-anchored signal peptide-CUB-EGF domain-containing proteins (SCUBE) gene family composed of three members was originally identified from endothelial cells (ECs). We recently showed that membrane SCUBE2 binds vascular endothelial growth factor (VEGF) and acts as a co-receptor for VEGF receptor 2 to modulate EC migration, proliferation, and tube formation during postnatal and tumour angiogenesis. However, whether these SCUBE genes cooperate in modulating VEGF signalling during embryonic vascular development remains unknown. To further dissect the genetic interactions of these scube genes, transcription activator-like effector nuclease-mediated genome editing was used to generate knockout (KO) alleles of each scube gene. No overt vascular phenotypes were seen in any single scube KO mutants because of compensation by other scube genes during zebrafish development. However, scube1 and scube2 double KO (DKO) severely impaired EC filopodia extensions, migration, and proliferation, thus disrupting proper vascular lumen formation during vasculogenesis and angiogenesis as well as development of the organ-specific intestinal vasculature. Further genetic, biochemical, and molecular analyses revealed that Scube1 and Scube2 might act cooperatively at the cell-surface receptor level to facilitate Vegfa signalling during zebrafish embryonic vascularization. We showed for the first time that cooperation between scube1 and scube2 is critical for proper regulation of angiogenic cell behaviours and formation of functional vessels during zebrafish embryonic development.

Endoplasmic reticulum stress and oncomir-associated chemotherapeutic drug resistance mechanisms in breast cancer tumors.

Breast cancer, as a heterogenous malign disease among the top five leading causes of cancer death worldwide, is defined as by far the most common malignancy in women. It contributes to 25% of all cancer-associated deaths after menopause. Breast cancer is categorized based on the expression levels of cell surface and intracellular steroid receptors [estrogen, progesterone receptors, and human epidermal growth factor receptor (HER2)], and the treatment approaches frequently include antiestrogen, aromatase inhibitors, and Herceptin. However, the management and prevention strategies due to adverse side effects stress the patients. The unsuccessful treatments cause to raise the drug levels, leading to excessive toxic effects on healthy cells, and the development of multidrug-resistance (MDR) in the tumor cells against chemotherapeutic agents. MDR initially causes the tumor cells to gain a metastatic character, and subsequently, the patients do not respond adequately to treatment. Endoplasmic reticulum (ER) stress is one of the most important mechanisms supporting MDR development. ER stress-mediated chemotherapeutic resistance is very common in aggressive tumors. The in vitro and in vivo experiments on breast tumors indicate that ER stress-activated protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)- activating transcription factor (ATF4) signal axis plays an important role in the survival of tumors and metastasis. Besides, ER stress-associated oncogenic microRNAs (miRNAs) induce chemoresistance in breast tumors. We aimed to have a look at the development of resistance mechanisms due to ER stress as well as the involvement of ER stress-associated miRNA regulation following the chemotherapeutic regimen in the human breast tumors. We also aimed to draw attention to potential molecular markers and therapeutic targets.

An effective polymeric nanocarrier that allows for active targeting and selective drug delivery in cell coculture systems.

In this manuscript, we report the development of a versatile, robust, and stable targeting nanocarrier for active delivery. This nanocarrier is based on bifunctionalized polymeric nanoparticles conjugated to a monoclonal antibody that allows for active targeting of either (i) a fluorophore for tracking or (ii) a drug for monitoring specific cell responses. This nanodevice can efficiently discriminate between cells in coculture based on the expression levels of cell surface receptors. As a proof of concept, we have demonstrated efficient delivery using a broadly established cell surface receptor as the target, the epidermal growth factor receptor (EGFR), which is overexpressed in several types of cancers. Additionally, a second validation of this nanodevice was successfully carried out using another cell surface receptor as the target, the cluster of differentiation 147 (CD147). Our results suggest that this versatile nanocarrier can be expanded to other cell receptors and bioactive cargoes, offering remarkable discrimination efficiency between cells with different expression levels of a specific marker. This work supports the ability of nanoplatforms to boost and improve the progress towards personalized medicine.

HBx increases EGFR expression by inhibiting miR129–5p function

Despite the efficient suppression of hepatitis B virus (HBV) replication by nucelos(t)ide analogs, HBV RNA expression usually continues even during nucleots(t)ide analog therapy because episomal covalently closed circular DNA (ccDNA), which is the template for HBV RNA transcription, cannot be eliminated. Here, we found that the common sequences of all HBV RNAs and that encoding the X protein (HBx) have similarities with the sequences of a host cellular microRNA (miRNA), miR129–5p. HBx inhibits miR129–5p function, resulting in increased expression of ZBTB20, a target gene of miR129–5p. ZBTB20 activates transcription and increases cell-surface epidermal growth factor receptor (EGFR) levels, promoting the cell growth rate, and this effect was reversed through ZBTB20 knockdown. mir129–5p levels in Ago2-containing complexes were reduced by expression of HBx, suggesting that the viral RNA sequestered miR129–5p from Ago2-containing complexes. These results indicate the possibility that HBV RNA may maintain pathogenicity even through nucleos(t)ide analog therapy.

The phenotype of NK-cells in the dynamics of the post-operative period in patients with peritonitis in depending on the outcome of the disease

Our study was aimed at investigating dynamic phenotype pattern of peripheral blood NK cells in patients with widespread purulent peritonitis (WPP) during postoperative period depending on disease outcome. A total of 48 patients aged 30–63 with acute surgical diseases and abdominal injuries complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period) as well as on day 7, 14 and 21 during postoperative period. 40 apparently healthy age-matched subjects were included in control group. Peripheral blood NK cell phenotyping was performed by using flow cytometry with directly immunofluorescently tagged antibodies. Mean fluorescence intensity was measured to estimate expression levels of NK cell surface receptors was measured. It was found that in patients with a favorable WPP outcome during preoperative period the percentage of mature NK cells was decreased that was restored by the end of the postoperative period (21 days post-surgery) due to elevated mature, cytotoxic and cytokine-producing NK cell subsets. In addition, percentage of CD11b-positive NK cell subsets was increased upon favorable outcome by the end of postoperative period as well as frequency of CD57-positive NK cells relative to the preoperative period. However, frequency of mature NK cells with unfavorable WPP outcome vs. control vs. favorable outcome was decreased during preoperative and entire postoperative period. Moreover, amount of cytotoxic NK cells was elevated during examination period upon unfavorable WPP outcome. Further, percentage of mature CD11b-positive NK cells in this patient cohort was decreased during preoperative period and post-surgery. Percentage of CD57-positive NK cells was decreased during entire postoperative period in patients with unfavorable vs. favorable outcome vs. control group. At the same time, patients with unfavorable outcome of this infectious-inflammatory disease were shown to display upregulated expression of CD28 and CD57 markers on NK cells. such features identified in phenotype of peripheral blood NK cells in patients with unfavorable WPP outcome reflect abnormal mechanisms in NK cell maturation and migration, which, in turn, determines disturbance in events regulating acute inflammatory reaction in WPP.

The myotubularin MTMR4 regulates phagosomal phosphatidylinositol 3-phosphate turnover and phagocytosis

Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain- and PX motif-containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated Mtmr4 silencing increased levels of cell-surface immunoglobulin receptors (i.e. Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and Mtmr4-specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with Mtmr4-specific siRNA were more resistant to Mycobacterium marinum-induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the Mtmr4-knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.

Quantitative proteomics reveals reduction of endocytic machinery components in gliomas

BackgroundGliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. MethodsWe performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry followed by bioinformatic analysis. FindingsVarious pathways were found to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. InterpretationWe propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. FundThis work was supported by Grants 316030-164105 (to P. Jenö), 31003A-162643 (to M. Spiess) and PP00P3-176974 (to G. Hutter) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel.

Synthesis and preclinical evaluation of the 177Lu-DOTA-PSMA(inhibitor)-Lys3-bombesin heterodimer designed as a radiotheranostic probe for prostate cancer.

Human tumors show intrinsic heterogeneity and changes in phenotype during disease progression, which implies different expression levels of cell surface receptors. The research on new heterodimeric lutetium-177 (Lu)-radiopharmaceuticals interacting with two different targets on tumor cells is a strategy for improvement of radiotheranostic performance. This study aimed to synthesize and characterize the Lu-DOTA-PSMA(inhibitor)-Lys-bombesin (Lu-DOTA-iPSMA-Lys-BN) heterodimer and to evaluate its potential to target prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPr) overexpressed in prostate cancer. The heterodimeric conjugate was synthesized and characterized by infrarred, mass, and H-NMR spectroscopies. The ligand was labeled with Lu and the radiochemical purity was assessed by radio-high-performance liquid chromatography. PSMA/GRPr affinity and the heterobivalent effect on cell viability were evaluated in LNCaP and PC3 prostate cancer cell lines. The biodistribution profile (3 and 96 h) was assessed in athymic mice with induced prostate tumors. Using pulmonary LNCaP (PSMA-positive) and PC3 (GRPr-negative) micrometastasis models, the influence of heterobivalency and affinity on tumor uptake was quantified (micro-SPECT/CT). Lu-iPSMA-BN (radiochemical purity>98%) showed specific recognition for PSMA and GRPr (IC50=5.62 and 3.49 nmol/l, respectively) with a significant decrease in cell viability (10.15% of cell viability in LNCaP and 40.10% in PC3 at 48 h), as well as high LNCaP and PC3 tumor uptake (5.21 and 3.21% ID/g at 96 h, respectively). Micro-SPECT/CT imaging showed the heterodimer ability to target the tumors (SUVmax of 1.93±0.30 and 1.76±0.10 in LNCaP and PC3, respectively), possibly influenced by the heterobivalent effect. Lu-DOTA-iPSMA-Lys-BN showed suitable affinity for PSMA and GRPr. The results warrant further preclinical studies to establish the Lu-radiotracer theranostic efficacy.

Electrochemiluminescence Investigation of Glucose Transporter 4 Expression at Skeletal Muscle Cells Surface Based on a Graphene Hydrogel Electrode

In situ detection of the expression level of cell-surface receptors has become a hotspot study in recent years. We propose in this manuscript a novel strategy for sensitive electrochemiluminescence (ECL) detection of glucose transporter 4 (GLUT4) on human skeletal muscle cells (HSMCs). Graphene hydrogel (GH) was selected to fabricate a permeable electrode with the purpose of overcoming the steric hindrance of cells on electrode, which leads to errors in the detection of cell-surface receptors. GLUT4 was labeled with carbon dots (CDs), which generate ECL emission at the interface between GH and cells, so about half the amount of GLUT4 expressed at the cell surface could be determined, which provided an accurate GLUT4 expression quantification. The prepared cytosensor exhibited good analytical performance for HSMC cells, ranging from 500 to 1.0 × 106 cells·mL-1, with a detection limit of 200 cells·mL-1. The average amount of GLUT4 per HSMC cell was calculated to be 1.88 × 105. Furthermore, GLUT4 on HSMC surface had a 2.3-fold increase under the action of insulin. This strategy is capable of evaluating the receptors on the cell surface, which may push the application of ECL for disease diagnosis.

Quantitative Proteomics Reveals Global Reduction of Endocytic Machinery Components in Gliomas

Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. We performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry and found various pathways to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. We propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. Funding: This work was supported by Grants 316030-164105 (P.J.), 31003A-162643 (M.S.) and PP00P3-176974 (G.H.) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel. Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: Biopsy samples were collected from patients after obtaining informed consent according to the guidelines of the local ethical committee (Ethikkommission Nordwestschweiz, #42/10).

Regulation of an Opioid Receptor Chaperone Protein, RTP4, by Morphine.

Signaling by classic analgesics, such as morphine, is governed primarily by the relative abundance of opioid receptors at the cell surface, and this is regulated by receptor delivery to, and retrieval from, the plasma membrane. Although retrieval mechanisms, such as receptor endocytosis, have been extensively investigated, fewer studies have explored mechanisms of receptor maturation and delivery to the plasma membrane. A previous study implicated receptor transporter proteins (RTPs) in the latter process. Since not much is known about regulation of RTP expression, we initiated studies examining the effect of chronic morphine administration on the levels of RTPs in the brain. Among the four RTPs, we detected selective and region-specific changes in RTP4 expression; RTP4 mRNA is significantly upregulated in the hypothalamus compared with other brain regions. We examined whether increased RTP4 expression impacted receptor protein levels and found a significant increase in the abundance of mu opioid receptors (MOPrs) but not other related G protein-coupled receptors (GPCRs, such as delta opioid, CB1 cannabinoid, or D2 dopamine receptors) in hypothalamic membranes from animals chronically treated with morphine. Next, we used a cell culture system to show that RTP4 expression is necessary and sufficient for regulating opioid receptor abundance at the cell surface. Interestingly, selective MOPr-mediated increase in RTP4 expression leads to increases in cell surface levels of MOPr-delta opioid receptor heteromers, and this increase is significantly attenuated by RTP4 small interfering RNA. Together, these results suggest that RTP4 expression is regulated by chronic morphine administration, and this, in turn, regulates opioid receptor cell surface levels and function.

PI5P4Kγ functions in DTX1-mediated Notch signaling

Notch signaling is an evolutionarily conserved pathway that is essential for development, where it controls processes ranging from cell differentiation to survival. Transport through endosomes is a critical step in regulating Notch signaling capacity, where the E3 ubiquitin ligase DTX1 is thought to control Notch1 intracellular transport decisions by direct receptor ubiquitination. However, how DTX1 regulates Notch1 transport within endosomes and the consequence of Notch1 ubiquitination by DTX1 remain unresolved. Here we demonstrate that DTX1 colocalizes with Notch1 on tubulovesicular recycling endosomes. We find that DTX1 silencing leads to enhanced Notch1 recycling from this compartment to the cell surface via a rab4a-mediated transport route. This, in turn, increases Notch1 cell-surface levels and enhances signaling. Surprisingly, we discovered that DTX1 depletion also elevates Notch1 activity mediated by a mutant form of the receptor that lacks lysine residues for ubiquitination, suggesting that DTX1 targets additional factors. Using an activity-based screen for ubiquitination targets, we identified multiple DTX1 substrates including PI5P4Kγ, a lipid kinase involved in PI(4,5)P2 production. Immunolocalization analysis reveals that PI5P4Kγ, like DTX1 and Notch1, is present on tubulovesicular recycling endosomes. However, in contrast to DTX1, Notch1 signaling is inhibited by pharmacological inactivation or siRNA depletion of PI5P4Kγ. Moreover, loss of PI5P4Kγ activity decreases Notch1 recycling rates and reduces receptor cell-surface levels. Collectively, these findings argue that PI5P4Kγ positively regulates the Notch pathway by promoting receptor recycling. Additionally, they support a model where DTX1 controls Notch1 endosomal sorting decisions by controlling PI5P4Kγ-mediated production of PI(4,5)P2.

Glycogen synthase kinase 3β inhibition enhances Notch1 recycling.

The Notch signaling pathway is essential throughout development and remains active into adulthood, where it performs a critical role in tissue homeostasis. The fact that defects in signaling can lead to malignancy illustrates the need to control Notch activity tightly. GSK3β is an established regulator of the Notch signaling pathway, although its mechanism of action remains unclear. Given the emerging role for GSK3β in receptor trafficking, we tested the idea that GSK3β controls signaling by regulating Notch transport. Consistent with published reports, we find that GSK3β inhibition enhances Notch1 signaling activity. Immunolocalization analysis reveals that Notch1 localization within a tubulovesicular compartment is altered when GSK3β activity is disrupted. We also find that receptor cell surface levels increase following acute GSK3β inhibition. This is followed by elevated Notch intra-cellular domain (NICD) production and a corresponding increase in signaling activity. Moreover, Notch transport assays reveal that receptor recycling rates increase when GSK3β activity is inhibited. Collectively, results presented here support a model where GSK3β regulates signaling by controlling postendocytic transport of Notch1. Given that GSK3β activity is suppressed following stimulation by multiple signal transduction pathways, our findings also suggest that cells can modulate Notch1 activity in response to extracellular signals by mobilizing Notch1 from endosomal stores.