Examining the effects of sodium ions on the binding of antagonists to dopamine D2 and D3 receptors

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo

IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies

Rab7A Is Required for Efficient Production of Infectious HIV-1

Retroviruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Rab proteins regulate specific steps in intracellular membrane trafficking by recruiting tethering, docking and fusion factors, as well as the actin- and microtubule-based motor proteins that facilitate vesicle traffic. Using virological tests and RNA interference targeting Rab proteins, we demonstrate that the late endosome-associated Rab7A is required for HIV-1 propagation. Analysis of the late steps of the HIV infection cycle shows that Rab7A regulates Env processing, the incorporation of mature Env glycoproteins into viral particles and HIV-1 infectivity. We also show that siRNA-mediated Rab7A depletion induces a BST2/Tetherin phenotype on HIV-1 release. BST2/Tetherin is a restriction factor that impedes HIV-1 release by tethering mature virus particles to the plasma membrane. Our results suggest that Rab7A contributes to the mechanism by which Vpu counteracts the restriction factor BST2/Tetherin and rescues HIV-1 release. Altogether, our results highlight new roles for a major regulator of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle

Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

From Wiley via Jisc Publications RouterHistory: received 2020-10-29, rev-recd 2021-04-27, accepted 2021-04-28, pub-electronic 2021-06-04Article version: VoRPublication status: PublishedFunder: Wellcome Trust; Grant(s): 107636/Z/15/Z, 210002/Z/17/ZFunder: UKRI | Biotechnology and Biological Sciences Research Council (BBSRC); Id: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/R015864/1, BB/M011208/1Funder: UKRI | Medical Research Council (MRC); Id: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/T016043/1Funder: Cancer Research UK (CRUK); Id: http://dx.doi.org/10.13039/501100000289; Grant(s): A27445Funder: NIHR Manchester Biomedical Research Centre; Grant(s): IS‐BRC‐1215‐20007Funder: Breast Cancer Now; Grant(s): MAN‐Q2‐Y4/5Abstract: Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine‐tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling‐dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR‐mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF‐mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers