scholarly journals Low-density-lipoprotein receptors in human fibroblasts are not degraded in lysosomes

1989 ◽  
Vol 262 (2) ◽  
pp. 681-683 ◽  
Author(s):  
L A F Casciola ◽  
K I Grant ◽  
W Gevers ◽  
G A Coetzee ◽  
D R van der Westhuyzen
Keyword(s):  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Receptor Internalization ◽  
Low Density ◽  
Human Skin Fibroblasts ◽  
Lipoprotein Receptors ◽  
Ldl Receptors ◽  
Endocytic Vesicles ◽  
Rate Limiting

The rate of degradation of low-density-lipoprotein (LDL) receptors was measured in cultured human skin fibroblasts by [35S]methionine pulse-chase experiments. The half-life of LDL receptors was unaltered by inclusion of LDL in the medium (t1/2 11 h). Neither lysosomotropic inhibitors (chloroquine or NH4Cl) nor leupeptin inhibited the rate of receptor degradation in the absence of ligand. In cells incubated at 18 degrees C to inhibit the delivery of internalized ligands from endocytic vesicles to lysosomes, receptor degradation continued, but at the expected rate of about six times lower than that at 37 degrees C. Mutant LDL receptors defective in internalization were degraded at the same rate as normal receptors, suggesting that receptor internalization and recycling are not required for basal turnover. We conclude that the rate-limiting steps for, and probably the whole pathway of, degradation of normal LDL receptors does not take place in lysosomes.

scholarly journals Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts.

1979 ◽  
Vol 83 (3) ◽  
pp. 588-594 ◽  
Author(s):  
H S Kruth ◽  
J Avigan ◽  
W Gamble ◽  
M Vaughan
Keyword(s):  
Cell Density ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Low Density ◽  
Cellular Lipid ◽  
Cholesterol Accumulation ◽  
Ldl Receptors ◽  
Ldl Binding ◽  
In Cells

The effect of cell density on low density lipoprotein (LDL) binding by cultured human skin fibroblasts was investigated. Bound LDL was visualized by indirect immunofluorescence. Cellular lipid and cholesterol were monitored by fluorescence in cells stained with phosphine 3R and filipin, respectively. LDL binding and lipid accumulation were compared in cells in stationary and exponentially growing cultures, in sparsely and densely plated cultures, in wounded and non-wounded areas of stationary cultures, and in stationary cultures with and without the addition of lipoprotein-deficient serum. We conclude that LDL binding and cholesterol accumulation induced by LDL are influenced by cell density. It appears that, compared to rapidly growing cells, quiescent (noncycling) human fibroblasts exhibit fewer functional LDL receptors.

Release of low density lipoprotein receptors from human fibroblasts or Hela cells by tryptic digestion

1978 ◽  
Vol 84 (2) ◽  
pp. 366-373 ◽  
Author(s):  
Nicola Di Ferrante ◽  
Patricia V. Donnelly ◽  
Daniela T. Di Ferrante ◽  
Salvatore Toma ◽  
Antonio M. Gotto
Keyword(s):  
Hela Cells ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Tryptic Digestion ◽  
Low Density ◽  
Lipoprotein Receptors

scholarly journals Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts.

1981 ◽  
Vol 90 (3) ◽  
pp. 595-604 ◽  
Author(s):  
L S Barak ◽  
W W Webb
Keyword(s):  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Internalization ◽  
Low Density ◽  
Receptor Complexes ◽  
Heparin Sulfate ◽  
Cultured Human Fibroblasts ◽  
Coa Reductase

The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory.

Occurrence of low density lipoprotein receptors within large pits on the surface of human fibroblasts as demonstrated by freeze-etching

1978 ◽  
Vol 113 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Lelio Orci ◽  
Jean-Louis Carpentier ◽  
A. Perrelet ◽  
Richard G.W. Anderson ◽  
Joseph L. Goldstein ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptors ◽  
Freeze Etching

scholarly journals Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells.

1981 ◽  
Vol 88 (2) ◽  
pp. 441-452 ◽  
Author(s):  
R G Anderson ◽  
M S Brown ◽  
J L Goldstein
Keyword(s):  
Cho Cells ◽  
High Efficiency ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Cell Types ◽  
Low Density ◽  
Coated Pits ◽  
Human Carcinoma ◽  
Ldl Receptors

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.

scholarly journals A mutation and an antibody that affect chemical cross-linking of low-density lipoprotein receptors on human fibroblasts

1993 ◽  
Vol 289 (2) ◽  
pp. 569-573 ◽  
Author(s):  
D D Patel ◽  
A K Soutar ◽  
B L Knight
Keyword(s):  
Tertiary Structure ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Surface Membrane ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cross Linking ◽  
Low Density ◽  
Ldl Receptors ◽  
Surface Receptors

Treatment of normal fibroblasts with the bifunctional cross-linking reagent DTSSP [3,3′-dithiobis(sulphosuccinimidylpropionate)] at 4 degrees C converted approximately 40% of the cell-surface low-density lipoprotein (LDL) receptors into a high-M(r) form, thought to represent receptor dimers. Preincubation of the cells with anti-(LDL receptor) monoclonal antibody 10A2 increased the proportion of surface receptors in the high-M(r) form after treatment with DTSSP at 4 degrees C to over 70%. Preincubation with LDL did not affect the proportion cross-linked, but prevented the increase produced by antibody 10A2. Cross-linking at 37 degrees C was less efficient than at 4 degrees C and was not affected by preincubation with antibody 10A2. Surface LDL receptors on fibroblasts from the homozygous familial hypercholesterolaemic subject MM were not cross-linked by DTSSP, confirming that the mutation had produced a change in the conformation of the receptor molecule. Taken together, the results suggest that normal LDL receptors on at least one region of the surface membrane may be loosely associated in some form of multimeric array which alters its alignment differently in response to antibody 10A2 and to cooling. Mutations that alter the tertiary structure of the receptors could affect LDL binding by disturbing the arrangement of the array.

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