Ligand-Induced Clustering of the LDL Receptor in the Absence of Coated Pits

1988 ◽  
Vol 46 ◽  
pp. 228-229
Author(s):  
Richard G. W. Anderson ◽  
William C. Donzell ◽  
Janet M. Larkin
Keyword(s):  
Cell Surface ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Cytoplasmic Tail ◽  
Receptor Clustering ◽  
Coated Pits ◽  
Remarkable Feature

A remarkable feature of the LDL receptor in human fibroblasts is that in the absence of ligand there is a preferential clustering of the receptor over clathrin-coated pits. The physiologic significance of this distribution was made apparent when it was discovered that certain mutations localized to the cytoplasmic tail of the LDL receptor resulted in the receptor being randomly distributed on the cell surface and unable to efficiently internalize LDL. These results raise the questions of what determinants in the coated pit are responsible for receptor clustering and whether the receptor is able to cluster in the absence of coated pits. Although the answer to the first question is still not known, we've been able to address the second question by utilizing a method for removing the coated pits in situ. This method depends upon the observation that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface.

scholarly journals The LDL Receptor Clustering Motif Interacts with the Clathrin Terminal Domain in a Reverse Turn Conformation

1998 ◽  
Vol 142 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Richard G. Kibbey ◽  
Josep Rizo ◽  
Lila M. Gierasch ◽  
Richard G.W. Anderson
Keyword(s):  
Nuclear Overhauser Effect ◽  
Ldl Receptor ◽  
Line Broadening ◽  
Wild Type ◽  
Receptor Clustering ◽  
Coated Pits ◽  
Overhauser Effect ◽  
Reverse Turn ◽  
Nuclear Overhauser ◽  
Terminal Domains

Previously the hexapeptide motif FXNPXY807 in the cytoplasmic tail of the LDL receptor was shown to be essential for clustering in clathrin-coated pits. We used nuclear magnetic resonance line-broadening and transferred nuclear Overhauser effect measurements to identify the molecule in the clathrin lattice that interacts with this hexapeptide, and determined the structure of the bound motif. The wild-type peptide bound in a single conformation with a reverse turn at residues NPVY. Tyr807Ser, a peptide that harbors a mutation that disrupts receptor clustering, displayed markedly reduced interactions. Clustering motif peptides interacted with clathrin cages assembled in the presence or absence of AP2, with recombinant clathrin terminal domains, but not with clathrin hubs. The identification of terminal domains as the primary site of interaction for FXNPXY807 suggests that adaptor molecules are not required for receptor-mediated endocytosis of LDL, and that at least two different tyrosine-based internalization motifs exist for clustering receptors in coated pits.

scholarly journals Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.

1990 ◽  
Vol 111 (2) ◽  
pp. 347-359 ◽  
Author(s):  
R K Pathak ◽  
M Yokode ◽  
R E Hammer ◽  
S L Hofmann ◽  
M S Brown ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transgenic Mice ◽  
Intestinal Epithelial Cells ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Intestinal Epithelial ◽  
Coated Pits ◽  
Ldl Receptors

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.

Abstract 18667: Lipoprotein(a) Catabolism and Apolipoprotein(a) Secretion is Regulated by Sortilin

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Rocco Romagnuolo ◽  
Matthew Gemin ◽  
Marlys Koschinsky
Keyword(s):  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Coated Pits ◽  
Direct Role ◽  
Lipoprotein A ◽  
Hepatic Cells ◽  
Apolipoprotein A ◽  
Ability To Act ◽  
Ldl Catabolism

Elevated levels of lipoprotein(a) (Lp(a)) in plasma have been identified as an independent, causal risk factor for coronary heart disease. Lp(a) consists of a low-density lipoprotein (LDL)-like particle whose apolipoproteinB-100 (apoB-100) moiety is covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). Recently, Lp(a) internalization by the LDL-receptor (LDLr) in hepatic cells and primary human fibroblasts has been shown to be regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9). However, Lp(a)/apo(a) internalization still occurs even with a defective LDLr or in the presence of PCSK9 (in fibroblasts and hepatic cells, respectively), indicating a role for receptors other than the LDLr in Lp(a) catabolism. Hepatic sortilin has been identified as a potential receptor mediating LDL catabolism as well as the regulation of apoB-100 secretion. Sortilin localizes to the Golgi apparatus where it mediates trafficking of specific bound ligands to the lysosome. Sortilin also localizes to clathrin-coated pits in the plasma membrane where it can act as an internalization receptor. We demonstrate in the current study that Lp(a), but not apo(a), internalization in hepatic cells is influenced by sortilin overexpression. In the presence of PCSK9, Lp(a) internalization greatly increases with sortilin overexpression compared to control. These results suggest that hepatic sortilin has the ability to act as a receptor for Lp(a) catabolism, through the LDL-like moiety, in a manner that is not dependent on the LDLr. Furthermore, Lp(a) internalization increases with sortilin overexpression in primary human fibroblasts with a defective LDLr, again emphasizing an LDLr-independent role for sortilin as a receptor for Lp(a). Interestingly, an increase in apo(a) secretion is observed with sortilin overexpression in hepatic cells. Removal of the carboxyl-terminal tail of sortilin results in an inability to promote the secretion of apo(a), indicating a direct role for the sorting motifs present in this region of sortilin for the regulation of apo(a) secretion. Taken together, these results indicate novel roles for sortilin in both Lp(a) catabolism and apo(a) secretion.

Platelet factor 4 binds to low-density lipoprotein receptors and disrupts the endocytic itinerary, resulting in retention of low-density lipoprotein on the cell surface

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3613-3622 ◽  
Author(s):  
Bruce S. Sachais ◽  
Alice Kuo ◽  
Taher Nassar ◽  
Jeanelle Morgan ◽  
Katalin Kariko ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Cultured Cells ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Platelet Factor 4 ◽  
Low Density ◽  
Coated Pits ◽  
Platelet Factor

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R–dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 μg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.

A mutation that impairs the ability of lipoprotein receptors to localise in coated pits on the cell surface of human fibroblasts

Nature ◽  
1977 ◽  
Vol 270 (5639) ◽  
pp. 695-699 ◽  
Author(s):  
Richard G. W. Anderson ◽  
Joseph L. Goldstein ◽  
Michael S. Brown
Keyword(s):  
Cell Surface ◽  
Human Fibroblasts ◽  
Coated Pits ◽  
Lipoprotein Receptors

scholarly journals Model System for Phenotypic Characterization of Sequence Variations in the LDL Receptor Gene

2006 ◽  
Vol 52 (8) ◽  
pp. 1469-1479 ◽  
Author(s):  
Trine Ranheim ◽  
Mari Ann Kulseth ◽  
Knut Erik Berge ◽  
Trond Paul Leren
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Ldl Receptor ◽  
Model System ◽  
Phenotypic Characterization ◽  
Sequence Variant ◽  
Ldlr Gene ◽  
Coated Pits ◽  
Sequence Variations

Abstract Background: Sequence variations in the LDL receptor (LDLR) gene cause defects of LDLR protein production and function through different molecular mechanisms. Here we describe a cell model system for the phenotypic characterization of sequence variations in the LDLR gene. Well-known sequence variations belonging to LDLR classes 2 to 5 (p.G565V, p.I161D, p.Y828C, and p.V429M) were studied in CHO and HepG2 cells. Methods: Expression of LDLR protein on the cell surface was detected by use of fluorescence-conjugated antibodies against the LDLR and the LDLR activity was measured by incubating the cells with fluorescently labeled and radiolabeled LDL. The intracellular locations of the LDLR mutants and wild-type were also investigated. Results: The class 2A p.G565V sequence variant exhibited an intracellular distribution of LDLR with no active receptors on the cell surface. Both the class 3 p.I161D and class 4 p.Y828C sequence variants gave surface staining but had a reduced ability to bind or internalize LDL, respectively. By determining the intracellular locations of the receptors we were able to visualize the accumulation of the class 5 p.V429M sequence variant in endosomes by means of a specific marker, as well as confirming that the class 4 p.Y828C variant was not localized in clathrin-coated pits. Flow cytometry allowed us quantitatively to determine the amount and activity of receptors. To confirm the results of binding and cell association of fluorescently labeled LDL analyzed by flow cytometry, assays using 125I-labeled LDL were performed. In addition to a useful and valid alternative to radiolabeled LDL, the unique properties of fluorescently labeled LDL allowed a variety of detection technologies to be used. Conclusions: This new approach enables phenotypic characterization of sequence variations in the LDLR gene. The assays developed may be valuable for confirming the pathogenicity of novel missense sequence variations found throughout the LDLR gene.

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

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