Early appearance of dispersed low density lipoprotein receptors on the fibroblast surface during recycling

We have examined the distributions of recycling low density lipoprotein receptors (LDL-Rs) as they emerge onto and cluster on the surfaces of cultured cells. Surface LDL-Rs were labeled with colloidal gold-LDL conjugates (AuLDL) and cells viewed as whole-mounts in the transmission electron microscope. The steady-state distribution of LDL-Rs on the cell surface, labeled with AuLDL at 4 degrees C, comprised ring-shaped clusters of receptors with dispersed receptors scattered amongst them. After 12 min of incubation at 37 degrees C, virtually all AuLDL probes were internalized. Electron microscopy of thin sections revealed clustered receptors in coated pits and the progressive accumulation of AuLDL in endosomes, multivesicular bodies and lysosomes. By initially blocking all surface LDL-Rs, either with unconjugated LDL or AuLDL of one size, the clustering behavior of newly emerged receptors which recycled to the cell surface was selectively visualized with an AuLDL probe of a second size over a defined time-course. Release of the blocking ligand during the time-course was found to be negligible. Newly appearing dispersed LDL-Rs were detected as early as 2 min and these were often concentrated at the cell margins. The newly labeled and preblocked LDL-Rs did not cocluster before 6 min. By 8 to 12 min, ring-shaped clusters of newly emerged receptors had formed and these were often seen associated with pre-blocked LDL-Rs. The clustering of LDL-Rs on the cell surface was independent of the presence of ligand, AuLDL. Our results indicate that LDL-Rs recycle to the cell surface where they form a dispersed population which gives rise to the ring-shaped clusters of cell surface LDL-Rs associated with coated pits.     

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

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.     

Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices

Previous studies have shown that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface and the receptor-mediated endocytosis of low density lipoprotein (LDL) is inhibited. We have now used the K+ depletion protocol to study several aspects of coated pit function. First, since coated pits rapidly form when K+-depleted fibroblasts are incubated in the presence of 10 mM KCl, we studied the sequence of assembly of coated pits as visualized in carbon-platinum replicas of inner membrane surfaces from cells that had been incubated in the presence of K+ for various times. New coated pits initially appeared as planar clathrin lattices that increased in size by the formation of polygons at the margin of the lattice. Once the lattice reached a critical size it invaginated to form coated vesicles. Second, we determined that LDL-ferritin can induce clustering of LDL receptors over noncoated membrane on the surface of K+-depleted fibroblasts; however, when these cells are subsequently incubated in the presence of K+, these clusters become associated with newly formed coated pits and are internalized. Finally, we determined that K+ depletion inhibits the assembly of coated pits, but that existing coated pits in K+-depleted cells are able to internalize LDL. These results suggest that the clathrin lattice of coated pits is actively involved in membrane shape change during endocytosis and that the structural proteins of the lattice are cyclically assembled and disassembled in the process.     

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

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.     

Effect of preferential insertion of LDL receptors near coated pits

Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10 plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37°C.     

Effect of preferential insertion of LDL receptors near coated pits

Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10, plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37 degrees C.     

Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.     

Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

Diffusion-limited forward rate constants in two dimensions. Application to the trapping of cell surface receptors by coated pits.

A variety of receptors are known to aggregate in specialized cell surface structures called coated pits, prior to being internalized when the coated pits close off. At 37 degrees C on human fibroblasts, as well as on other cell types, a recycling process maintains a constant number of coated pits on the cell surface. In this paper, we explore implications for receptor aggregation and internalization of the two types of recycling models that have been proposed for the maintenance of the coated pit concentration. In one model, coated pits alternate between accessible and inaccessible states at fixed locations on the cell surface, while in the other model, coated pits recycle to random locations on the cell surface. We consider receptors that are randomly inserted in the membrane, move by pure diffusion with diffusion coefficient D, and are instantly and irreversibly trapped when they reach a coated pit boundary (the diffusion limit). For such receptors, we calculate for each of the two models: the mean time tau to reach a coated pit, the forward rate constant k+ for the interaction of a receptor with a coated pit, and the fraction phi of receptors aggregated in coated pits. We show that for the parameters that characterize coated pits on human fibroblasts, the way in which coated pits return to the surface has a negligible effect on the values of tau, k+, and phi for mobile receptors, D greater than or equal to 1.0 X 10(-11) cm2/s, but has a substantial effect for "immobile" receptors, D much less than 1 X 10(-11) cm2/s. We present numerical examples to show that it may be possible to distinguish between these models if one can monitor slowly diffusing receptors (D less than 1 X 10(-11) cm2/s) on cells whose coated pits have relatively short lifetimes (less than or equal to 1 min). Finally, we show that for the low-density lipoprotein (LDL) receptor on human fibroblasts (D = 4.5 X 10(-11) cm2/s), the predicted and observed values of K+ and phi are in close agreement. Therefore, even for slowly diffusing LDL receptor, unaided diffusion as the transport mechanism of receptors to coated pits is consistent with measured rates of LDL internalization.     

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation

Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K+ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273-285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679). Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson, 1986. J. Cell Biol. 103:2619-2627), we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freeze-etched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages". At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37 degrees C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K+ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits.     

Hepatic binding and internalization of low density lipoprotein-gold conjugates in rats treated with 17 alpha-ethinylestradiol

Receptor-mediated hepatic uptake of low density lipoproteins (LDL) conjugated to colloidal gold was studied by perfusion of livers from rats treated for 5 d with 17 alpha-ethinylestradiol. Estrogen treatment resulted in a marked decrease in serum lipid and lipoprotein concentrations. After 15 min of perfusion the conjugate was bound to the hepatic microvilli of both control and estrogen-treated rats; the estrogen-treated rats showed an 8- to 11-fold greater number of membrane-bound conjugates. The conjugates were bound to the membrane receptor by the LDL particle because the gold granules were regularly displaced from the membrane by 20 +/- 3.2 nm, the diameter of LDL. Internalization of the conjugate, evident by gold particles in multivesicular bodies, occurred at coated pits at the base of the microvillus where coated vesicles containing a single gold-LDL conjugate were released. After 1 h of perfusion, the livers from the estrogen-treated rats showed all phases of endocytosis and incorporation into multivesicular bodies of the conjugate. After 2 h of perfusion, there was congregation of gold-labeled lysosomes near the bile canaliculi. Gold-LDL conjugates were also observed to bind and be internalized by Kupffer cells and sinusoidal endothelium. These findings indicate that estrogen treatment induces hepatic receptors for LDL. The catabolic pathway of binding and endocytosis of the conjugate is similar to that seen in fibroblasts, although slower. Because gold-LDL conjugates were also present in the Kupffer and endothelial cells, the uptake of LDL by the liver involves the participation of more than a single cell type.     

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.     

Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.     

The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits

ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.     

Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts

In cultured human fibroblasts, each LDL receptor mediates the internalization of approximately 100 particles of LDL every 20 hr. We provide evidence that this reutilization of LDL receptors involves the recycling of receptors into and out of the cell and that the carboxylic ionophore monensin blocks the return of the receptors to the surface. In the presence of monensin and LDL, 75% of the receptors disappeared from the cell surface within 15 min and more than 90% disappeared within 60 min. The receptors that left the surface were trapped intracellularly within perinuclear vacuoles, as visualized by indirect immunofluorescence with the use of LDL, monensin caused about 50% of the receptors to be trapped intracellularly within 15 min. The receptors that remained on the surface after monensin treatment could be trapped within the cell if LDL was added subsequently in the continued presence of monensin. Monensin did not decrease surface LDL receptors in fibroblasts from a patient (J.D.) with the internalization-defective form of familial hypercholesterolemia. In these mutant cells, LDL receptors are not localized to coated pits. The current data are interpreted to indicate that: in normal fibroblasts about 50% of surface LDL receptors absence of LDL; the remaining 50% of surface receptors can be induced to recycle by the presence of LDL; and monensin interrupts this recycling by preventing the receptor from returning to the surface, thereby causing the receptors to accumulate within the cell.     

Native and acetylated low density lipoprotein metabolism in proliferating and quiescent bovine endothelial cells in culture

Lipoprotein binding and metabolism in actively dividing (sparse) and quiescent (confluent) bovine aortic endothelial cells (EC) were compared quantitatively using 125I-labelled lipoproteins. The amounts of receptor-bound low density lipoproteins (LDL) decreased five- to ten-fold as the cultures progressed from sparse to confluent morphology. High affinity receptor-bound LDL levels were extremely low in confluent EC and accounted for the inability of confluent EC to internalize and degrade significant amounts of LDL. Conversely, the amounts of acetylated LDL (acLDL) bound and degraded via distinct sites increased at least five-fold during EC growth to confluence. LDL binding and metabolism in individual cells was assessed by fluorescence microscopy using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled lipoproteins or fluorescein-conjugated antibodies. LDL and acLDL bound to the surfaces of sparse EC, at either 4 degrees or 37 degrees C, in a random distribution of fine punctate foci, contrary to a previous report. EC therefore appear to resemble fibroblasts in their distribution of surface LDL receptors. No binding or uptake of LDL was seen in confluent EC. Patterns of acLDL binding and uptake in confluent EC resembled those of LDL in sparse EC. Intracellular LDL and acLDL occurred as perinuclear accumulations of large fluorescent foci in sparse EC. Regeneration experiments were carried out in artificially wounded confluent cultures and renewed LDL receptor activity was shown in actively-dividing cells which had migrated into the "wounded" areas. We conclude that quiescent endothelial cells metabolize little LDL via the LDL-receptor pathway due to a drastically reduced number of receptors in confluent cells. This contrasts with the ability of confluent cells to metabolize relatively large amounts of acLDL via a receptor-mediated mechanism.     

The effect of diffusion on the trapping of membrane-bound receptors by localized coated pits

Localized coated pits are considered in the primary steps of receptor-mediated endocytosis. According to the pit reinsertion mechanism, we have modified our previous kinetic model and studied the effect of diffusion on the trapping rate constant (k+). Using experimental data for low density lipoprotein (LDL) receptors on fibroblast cells, we found that the binding of receptors to coated pits is not totally diffusion controlled. For example, the process is less than 78% diffusion controlled if receptors are not allowed to escape the coated pits. However, due to the large uncertainties in the experimental parameters, a diffusion-controlled process cannot be ruled out. The greatest differences between localized and random reinsertion were found when the escaping rate constant (k-) is much greater than the rate constant for invagination of the pits (lambda 1). Under this condition, k+ for localized reinsertion is no less than 39% diffusion controlled, while k+ for random reinsertion shows no diffusion effect at all.     

Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain

Certain mutant alleles at the low density lipoprotein (LDL) receptor locus produce receptors that bind LDL normally, but fail to cluster in coated pits and therefore cannot transport LDL into cells. We prepared genomic DNA libraries from cells of two individuals with this phenotype (internalization-defective familial hypercholesterolemia) and isolated the segment of the gene encoding the COOH-terminal cytoplasmic domain of the receptor. One mutant gene contains a single base substitution that changes a tryptophan codon (TGG) to a termination codon (TGA). This produces a receptor with only two amino acids in the cytoplasmic domain. The second mutant gene contains a four-base duplication, producing a frameshift that alters the reading frame. The cytoplasmic tail of this receptor has six of the normal amino acids plus eight additional amino acids. These data suggest that the signal for targeting the LDL receptor to coated pits resides in the cytoplasmic domain of the molecule.     

The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors

Genomic DNA encompassing the terminal exons of the gene for the low density lipoprotein (LDL) receptor was isolated from J.D., a patient with familial hypercholesterolemia whose receptor fails to cluster in coated pits. The DNA sequence revealed a substitution of a cysteine codon for a tyrosine codon at residue 807 in the cytoplasmic domain of the receptor. We reproduced this substitution through oligonucleotide-directed mutagenesis of the normal human receptor cDNA. Upon transfection into receptor-deficient hamster cells, the cDNA specified a receptor that bound LDL normally, but entered the cell slowly. Electron microscopy showed that this receptor was distributed diffusely over the cell surface, whereas the receptor produced by the normal cDNA was concentrated in coated pits. These results support the hypothesis that cytoplasmic domains direct receptors to coated pits, thereby determining the high rate of receptor internalization in animal cells.