Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Imaging LDL receptor oligomerization during endocytosis using a co-internalization assay.

ABSTRACT: Methods to probe receptor oligomerization are useful to understand the molecular mechanisms of receptor signaling. Here we report a fluorescence imaging method to determine receptor oligomerization state in living cells during endocytic internalization. The wild-type receptor is co-expressed with an internalization-defective mutant, and the internalization kinetics of each are independently monitored. If the receptor internalizes as an oligomer, then the wild-type and mutant isoforms will mutually influence each others' trafficking properties, causing co-internalization of the mutant or co-retention of the wild-type at the cell surface. Using this approach, we found that the low density lipoprotein (LDL) receptor internalizes as an oligomer into cells, both in the presence and absence of LDL ligand. The internalization kinetics of the wild-type receptor are not changed by LDL binding. We also found that the oligomerization domain of the LDL receptor is located in its cytoplasmic tail.

SUBMITTER: Zou P

PROVIDER: S-EPMC3078185 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Imaging LDL receptor oligomerization during endocytosis using a co-internalization assay.

Zou Peng P Ting Alice Y AY

ACS chemical biology 20110114 4

Methods to probe receptor oligomerization are useful to understand the molecular mechanisms of receptor signaling. Here we report a fluorescence imaging method to determine receptor oligomerization state in living cells during endocytic internalization. The wild-type receptor is co-expressed with an internalization-defective mutant, and the internalization kinetics of each are independently monitored. If the receptor internalizes as an oligomer, then the wild-type and mutant isoforms will mutual ...[more]

PMID: 21194239

Similar Datasets

Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor.

Project description:The internalization signals of several constitutively recycling receptors have recently been identified as regions of four or six amino acids that include an aromatic residue, usually tyrosine. Here, we show that transplanted signals from the low density lipoprotein receptor (LDLR) and cation-independent mannose-6-phosphate receptor (Man-6-PR) promote rapid internalization of the transferrin receptor (TR), directly establishing that recognition signals are interchangeable, self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. We also show that the chemical and spatial patterns of critical residues in both four- and six-residue internalization motifs are consistent with a tight turn structure. A six-residue LDLR signal is needed for activity in TR, suggesting that an amino-terminal aromatic side chain is obligatory. In contrast, the carboxy-terminal aromatic side chain in the TR signal can be replaced by a large hydrophobic residue. Thus, internalization signals apparently require an aromatic amino-terminal residue and either an aromatic or large hydrophobic carboxy-terminal residue rather than a conserved tyrosine per se. Consistent with this conclusion, predicted internalization signals from the poly-Ig receptor, YSAF, and asialoglycoprotein receptor (ASGPR) subunit H1, YQDL, also promote internalization of TR.

| S-EPMC453049 | biostudies-other

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging.

Project description:Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii) occurs independently of endocytic vesicle trafficking along microtubules, and (iv) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.

| S-EPMC1564263 | biostudies-literature

IRGM1 regulates oxidized LDL uptake by macrophage via actin-dependent receptor internalization during atherosclerosis.

Project description:Macrophage derived foam cells are actively involved in the initial phase of atherosclerosis. Uptake of modified lipoprotein such as oxidized LDL (oxLDL) is a critical step for foam cell formation. CD36 is the major receptor mediating oxLDL uptake by macrophage. However, the molecular mechanism underlying CD36 mediated oxLDL uptake remains unclear. Here we reported that IRGM1 (IRGM in human), a member of immunity-related small GTPase family, is essential for the actin-dependent CD36 mediated oxLDL uptake by macrophage. IRGM/IRGM1 was highly expressed by macrophage around the atherosclerotic plaque and was up-regulated by oxLDL both in vitro and in vivo. Moreover loss of IRGM/IRMG1 significantly decreased oxLDL uptake in both mouse and human. Furthermore, the IRGM1 knock-out mice displayed impaired CD36 internalization in macrophage, which was associated with the deficiency of F-actin polymerization. These results revealed a novel function of IRGM1 in regulating oxLDL uptake by macrophage during atherosclerosis.

| S-EPMC3659323 | biostudies-other

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization.

Project description:The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ?40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.

| S-EPMC2998144 | biostudies-other

Antibody/Ligand-Target Receptor Internalization Assay Protocol Using Fresh Human or Murine Tumor Ex Vivo Samples.

Project description:We describe an ex vivo EGF ligand internalization assay using fresh patient tumor biopsies to determine how antigen targets will be trafficked before patients receive mAb treatment. This protocol facilitates a sensitive and reproducible indication as to mAbs surface retention times during treatment. EGF uptake protocols can also be used to analyze EGFR heterogeneity and localization of EGFR in both tumor and xenograft tissue. The technology can be adapted to analyze other receptors such as PD-L1 for which methods are provided. For complete details on the use and execution of this protocol, please refer to Joseph et al. (2019) and Chew et al. (2020).

| S-EPMC7580087 | biostudies-literature

Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

Project description:CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (?2R) radioligands, [125I]RHM-4 or [3H]DTG, but a reduction in the receptor affinity of both radioligands was observed. TMEM97 KO resulted in a complete loss of binding of [125I]RHM-4 and a significant reduction in binding of [3H]DTG. TMEM97 KO and PGRMC1 KO resulted in an equal reduction in the rate of uptake of fluorescently-tagged or 3H-labeled LDL, and knocking out both proteins did not result in a further rate of reduction of LDL uptake. Confocal microscopy and Proximity Ligation Assay studies indicated a clear co-localization of LDLR, PGRMC1 and TMEM97. These data indicate that the formation of a ternary complex of LDLR-PGRMC1-TMEM97 is necessary for the rapid internalization of LDL by LDLR.

| S-EPMC6238005 | biostudies-literature

Endocytosis by macrophages: Interplay of macrophage scavenger receptor-1 and LDL receptor-related protein-1

Project description:Multiple receptors may mediate the cellular uptake of a single protein and thereby affect the plasma level of the involved protein. In case of Von Willebrand Factor (VWF) these receptors include LDL receptor-related protein-1 (LRP-1), Macrophage scavenger receptor-1 (MSR-1, SR-AI or CD204), the Macrophage Galactose-type lectin (CLEC10A, MGL or CD301), Siglec-5 and the Asialoglycoprotein receptor (ASGPR).1 In the present study, we aimed to gain insight into the interplay of multiple receptors to the cellular internalization of a single ligand like VWF. The macrophages in the liver and spleen have been reported to contribute considerably to the cellular uptake of VWF. Previously, we have shown that also human monocyte-derived macrophages (MDMs) internalize VWF via a mechanism that depends on LRP-1.5 We now analyzed the cell surface proteome of MDMs using mass spectrometry analysis to identify putative other VWF clearance receptors on MDMs. We found that MDMs contain LRP-1 and MSR-1, and we identified 1 peptide that is shared by Siglec-5 and Siglec-14. The estimated copy numbers of these receptors revealed a markedly higher expression of LRP-1 and MSR-1 compared to Siglec-5/14. We therefore focused our study on the possible dual mechanism by which LRP-1 and MSR-1 may cooperate in the cellular uptake of VWF by MDMs and conducted a range of functional studies. Based on the data presented in this study, we propose the following model to explain the relationship between the two abundant VWF receptors on MDMs. Both LRP-1 and MSR-1 associate with regions in the D’D3A1 region of VWF, thereby initiating two endocytic pathways that are both regulated by LRP-1. The first pathway follows a direct association of the VWF A1 domain to LRP-1. In the second pathway, VWF interacts to MSR-1 via regions in the D’D3 assembly, which subsequently associates to LRP-1 for endocytosis.

2019-07-08 | PXD011490 | Pride

PET imaging of dopamine-D2 receptor internalization in schizophrenia.

Project description:Recent genetic, molecular and post-mortem studies suggest impaired dopamine (DA)-D2 receptor (D2R) trafficking in patients with schizophrenia (SZ). Imaging and preclinical studies have shown agonist-induced D2R internalization can be imaged with positron emission tomography (PET) using D2R radiotracers combined with psychostimulant challenge. This is feasible if radiotracer binding is measured when postchallenge DA levels have returned to baseline, following the initial competition phase between DA and radiotracer for binding to D2R. Here we used 'late-phase' imaging after challenge to test the hypothesis that impaired D2R internalization in SZ leads to blunted late-phase displacement, or a faster return to baseline, in patients compared with healthy controls (HCs). We imaged 10 patients with SZ and 9 HCs with PET and [11C]raclopride at baseline and two times (3-5 and 6-10?h) following 0.5?mg?kg-1 dextroamphetamine. We measured binding potential relative to non-displaceable compartment (BPND) and derived percent reduction from baseline (?BPND) for each postamphetamine scan. To test the hypothesis that time course of return of striatal BPND to baseline differed between SZ and HCs, we implemented a linear model with ?BPND as dependent variable, time after amphetamine as repeated measure and time after amphetamine and diagnostic group as fixed effects. Neither diagnostic group nor interaction of diagnostic group-by-time after amphetamine significantly affected striatal ?BPND (F=1.38, P=0.26; F=0.51, P=0.61). These results show similar pattern of return of BPND to baseline as a function of time in patients with SZ and HC, suggesting that striatal D2R internalization as measured by our imaging paradigm is normal in patients with SZ.

| S-EPMC5690884 | biostudies-literature

Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis.

Project description:Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor class-A protein that is expressed on the cell surface of macrophages. MARCO mediates binding and ingestion of unopsonized environmental particles, including nano-sized materials. Exosomes are cell-derived, nano-sized vesicles (40-150 nm) that can contain lipids, RNA, DNA, and various proteins. Exosomes play an essential role in cell-to-cell communication via body fluids. However, mechanisms for the recognition and internalization of exosomes by recipient cells remain poorly characterized. In this study, cellular association of serum-derived fluorescent exosomes and 20-nm fluorescent nanoparticles (positive control) was compared between MARCO-expressing (CHO-MARCO) and control (CHO-CT) CHO-K1 cells to examine whether MARCO expression by recipient cells mediates the cellular uptake of exosomes and environmental nanoparticles. Fluorescence microscopic studies and quantitative analyses revealed that the cellular associations of both exosomes and 20-nm nanoparticles were greater in CHO-MARCO cells than in CHO-CT cells. Exosomes and nanoparticles colocalized with green fluorescent protein (GFP)-MARCO in cells transfected with GFP-MARCO-encoding constructs . Furthermore, inhibitory studies showed that actin reorganization and dynamin are involved in the MARCO-mediated cellular internalization of exosomes. These results indicated that MARCO plays a role in the uptake of exosomes.

| S-EPMC7733512 | biostudies-literature

Ajuba receptor mediates the internalization of tumor-secreted GRP78 into macrophages through different endocytosis pathways.

Project description:Glucose-regulated protein 78 (GRP78), an ER chaperone, is overexpressed in cancer cells. Solid tumor cells can secrete GRP78 that can promote tumor angiogenesis, differentiation of bone marrow-derived mesenchymal stem cells, tumor cell proliferation and polarization of tumor-associated macrophages. However, the mechanism by which GRP78 functions as a tumor promoter either by staying on the membrane to stimulate intracellular signals or directly entering into cytosolic remains unknown. Here, we reported that an endotoxin-free His-GRP78 protein was purified in vitro that simulates original secreted GRP78. Through analyzing GRP78 concentration in serum samples from 32 colon cancer patients, 40 nM His-GRP78 was selected as an optimized dose to treat cells. Biochemical analysis revealed that secreted GRP78 was able to enter into RAW264.7 and THP-1 cells directly rather than stay on the plasma membrane to transfer signals. Further studies showed that GRP78 internalization was endocytosis-dependent, and both phagocytosis and clathrin, caveolin-1 and micropinocytosis-mediated endocytosis pathways contributed to internalization of secreted GRP78 into cells. Mechanistically, Ajuba is able to interact with GRP78. Ablation of Ajuba suppressed the internalization of secreted GRP78 into cells, indicating that Ajuba was responsible for internalization of secreted GRP78 into RAW264.7. Furthermore, we observed that internalized GRP78 could entered into the mitochondrion and endoplasmic reticulum, which provided a suitable place and enough time for GRP78 to function in molecular and cellular processes. Together, these results reveal a novel mechanism by which secreted GRP78 internalizes into macrophages in the tumor microenvironment, which provides a potential target for drug development.

| S-EPMC5884641 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data