Binding, internalization, and intracellular localization of interleukin-1 beta in human diploid fibroblasts

This study demonstrates internalization of interleukin-1 (IL-1) via its cell surface receptor on human diploid fibroblasts and shows intracellular localization of IL-1 beta. Binding experiments at 8 degrees C using confluent fibroblast monolayers revealed 5,000-15,000 IL-1 receptors/cell that bound both IL-1 alpha and IL-1 beta. Incubation of monolayers with 125I-IL-1 beta (10(-9) M) at 8 degrees C and then at 37 degrees C for various times up to 8 h revealed a t1/2 for internalization of receptor-bound IL-1 beta of about 1.5 h. In addition, it was shown that IL-1 beta internalized via receptors was undegraded and retained binding activity. Electron microscopic autoradiography of monolayers incubated with 125I-IL-1 beta, as above, showed a progressive increase in the ratio of cytoplasmic to cell surface-associated grains. Grains at the cell surface were primarily localized at cell processes or attachment sites, frequently close to intra- and extracellular filamentous material. During incubation at 37 degrees C, most grains were free in the cytoplasm, with few present in lysosomes or vesicles. After 1 h, approximately 15% of the grains were over nuclei. Control cultures incubated at 37 degrees C with 125I-IL-1 beta and 100-fold excess unlabeled IL-1 beta showed increased uptake of label into lysosomes and little into nuclei. This study shows that IL-1 receptors are primarily located at fibroblast processes and that receptor-mediated internalization of the ligand is slow. Nuclear localization apparently requires IL-1 receptor-specific internalization of IL-1 beta, suggesting a possible role for this process in eliciting the IL-1 signal.     

IL-1 and its receptor are translocated to the nucleus

The internalization and intracellular transport of IL-1 and its receptor were examined in the murine T cell line EL-4. For 4 h after internalization intracellular 125I-IL-1 alpha remains bound to its receptor without degradation. Electron microscope autoradiography demonstrates that internalized IL-1 accumulates in purified nuclei. The IL-1 extracted from these nuclei is still bound to receptor. As no receptors for IL-1 were detected in untreated nuclei, these results suggest IL-1 driven translocation of the cell surface IL-1R complex to the nucleus. IL-1R internalization was correlated with IL-1 signal transduction events required to induce growth factor production from several subclones of EL-4 cells. The subsequent transport of the internalized IL-1R complex to the nucleus suggests the possibility for a nuclear site for IL-1R signaling.     

Acute upregulation of interleukin-1 receptor by ligand.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.     

Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction.

The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor.     

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization.

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.     

Interleukin-1 receptor-like proteins synthesized by translation, in vitro, of immunopurified polysomal messenger RNA

Interleukin-1 (IL-1) receptors can be solubilized from murine cell surfaces and immunoprecipitated with a xenogeneic rat antiserum raised in this laboratory. We demonstrated first that this antiserum contains antibodies directed against IL-1 receptors. We have now successfully used this antiserum as a reagent to immunopurify polysomes along with their messenger RNA from a murine leukemic cell line known to express relatively high levels of IL-1 receptors. The immunoselected mRNA was translated into proteins in vitro. The translation products contained an IL-1 binding protein which could specifically bind to immobilized IL-1 but not to other immobilized ligands such as interleukin-2 or tumor necrosis factor-alpha. The translation products which bound to IL-1 could be acid-eluted from the immobilized ligand, and the proteins released could still specifically bind to IL-1 in a receptor-ligand binding reaction. The eluted IL-1 binding proteins, as well as soluble receptor-ligand complexes derived from them, could also be immunoprecipitated with the xenogeneic rat antiserum. The xenogeneic rat antiserum could, furthermore, immunoprecipitate the IL-1 binding proteins from the translated products before ligand was added. The residual translated products no longer interacted with IL-1. We conclude that our antiserum contains antibodies that recognize determinants expressed on the following proteins: on nascent chains of IL-1 binding proteins; on soluble translated IL-1 binding proteins; on soluble complexes of IL-1 binding proteins that had been cross-linked with IL-1 ligand; and on cell surface-associated IL-1 receptors. The translated and unprocessed IL-1 binding proteins have a molecular mass of approximately 52,000-56,000 daltons.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells

Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCmu.     

Two molecular forms of the human interferon-gamma receptor. Ligand binding, internalization, and down-regulation

The receptors for human interferon-gamma (IFN-gamma) on peripheral blood monocytes and various cells of nonhematopoietic origin were thoroughly characterized and compared. The receptors of all cell types exhibited a similar affinity for IFN-gamma (Kd approximately 1 x 10(-10) M), and in all cases receptor-mediated endocytosis and ligand degradation were demonstrated. However, the receptors differed in their molecular weights (95,000 in HeLa cells and 140,000 in monocytes, assuming a 1:1 ligand to receptor ratio) as concluded from experiments of cross-linking to 125I-IFN-gamma. Lower molecular weight species were obtained as well, particularly in monocytes. Such species could represent either degradation products or subunit structures. The monocyte and HeLa receptor responded differently to an excess of ligand. A significant receptor down-regulation was observed when monocytes were incubated with an excess of 125I-IFN-gamma, whereas no such down-regulation was observed in HeLa cells or in normal fibroblasts. This differential response was observed both in the presence or in the absence of a protein synthesis inhibitor. The receptor on monocytes was found to be acid-labile whereas that on HeLa cells was resistant to acid treatment. These and additional experiments indicate that the monocyte receptor is inactivated following internalization, whereas the HeLa receptor retains its structure and recycles back to the cell surface. The difference in the properties and fate of these two receptor subtypes is probably related to the differential functions of IFN-gamma in various cell types.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

Phorbol ester induces phosphorylation of the 80 kilodalton murine interleukin 1 receptor at a single threonine residue.

The cytoplasmic domains of some cell surface receptors become phosphorylated in cells treated with phorbol esters. The present study was undertaken in order to determine whether this is also true of the 80 kDa interleukin 1 receptor (IL1R). Recombinant murine IL1R, transfected into chinese hamster ovary (CHO) cells or murine fibroblasts, was immunoprecipitated from [32P]orthophosphate-labelled cells. IL1R phosphorylation was only detected in cells pretreated with phorbol 12-myristate 13-acetate (PMA) and occurred solely on phosphothreonine. In contrast to a previous report, little or no IL1R phosphorylation occurred in response to IL1. By using a truncated receptor and receptors in which threonine residues were changed to alanines, we established that Thr537, near the carboxy-terminus, is the major site of PMA-induced phosphorylation. The human IL1R has a different sequence at this locus, and is apparently not phosphorylated. Binding studies showed that PMA-induced phosphorylation had no discernible effect on ligand binding or internalization.     

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.     

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.     

Extremely rapid endocytosis mediated by the mannose receptor of sinusoidal endothelial rat liver cells.

Isolated sinusoidal endothelial rat liver cells (EC) in suspension bound and internalized ovalbumin, a mannose-terminated glycoprotein, in a saturable manner. The binding and uptake were Ca2+-dependent and were effectively inhibited by alpha-methyl mannoside and yeast mannan, but not by galactose or asialoglycoproteins. This corresponds to the binding specificity described for the mannose receptor of macrophages and non-parenchymal liver cells. Binding studies indicated a surface pool of 20,000-25,000 mannose receptors per cell, with a dissociation constant of 6 x 10(-8) M. Uptake and degradation of ovalbumin by isolated EC were inhibited by weak bases and ionophores which inhibit acidification of endocytic vesicles and dissociation of receptor-ligand complexes. Cycloheximide had no effect on uptake or degradation. Degradation, but not uptake, was inhibited by leupeptin. We conclude that ovalbumin dissociates from the mannose receptors in the endosomal compartment and the receptors are recycled to the cell surface, while the ovalbumin is directed to the lysosomes for degradation. A fraction of the internalized ovalbumin was recycled intact to the cell surface and escaped degradation (retroendocytosis). The rate of internalization of ovalbumin by isolated EC was very fast, with a Ke (endocytotic rate constant) of 4.12 min-1, which corresponds to a half-life of 10 s for the surface pool of receptor-ligand complexes. To our knowledge, this is the highest Ke reported for a receptor-mediated endocytosis system.     

Interleukin-1 stimulates diacylglycerol production in T lymphocytes by a novel mechanism

We have investigated the biochemical mechanism by which interleukin-1 (IL-1) serves as a comitogen with agents that directly activate the antigen receptor in T lymphocytes. We have studied the human T cell line Jurkat, which can be stimulated to produce Interleukin-2 by treatment with antibodies that bind to the CD3-antigen receptor complex and hence represents a model system for T cell activation. Using highly purified, recombinant human IL-1, we show that IL-1 stimulates rapid diacylglycerol and phosphorylcholine production from phosphatidylcholine (PC) in the absence of phosphatidylinositol turnover in Jurkat cells. This effect is also observed in peripheral blood T cells and a murine T cell line. The EC50 for IL-1 was 28 fM, and PC hydrolysis was detectable within 5 sec at 37 degrees C. The murine cell line had typical high-affinity IL-1 receptors (kd = 7 X 10(-11) M). However, we were unable to detect IL-1 binding to Jurkat cells. This reaction occurs via a novel mechanism and may explain the comitogenic activity of IL-1 in T lymphocyte activation as well as many of the pleiotropic biologic effects of this cytokine.     

The interleukin 1 receptor. Dynamics of interleukin 1 binding and internalization in T cells and fibroblasts

In this study, we have demonstrated that a murine T cell lymphoma, EL 4, and a murine fibroblast cell line, Swiss 3T3, possess a single class of high affinity interleukin 1 (IL 1) receptors that exist in a dynamic state of equilibrium that is influenced by IL 1. In the absence of IL 1, the IL 1 receptor appears to turnover with a t1/2 of approximately 11 hr. However, when cells are incubated in the presence of IL 1, the IL 1 receptor undergoes extensive ligand-induced down-regulation. IL 1 itself is internalized at 37 degrees C; 50% of the surface-bound IL 1 is internalized in 60 to 120 min. IL 1 does not undergo degradation for at least 6 hr after internalization. By using electron microscopy and autoradiography, we observed several important features of the internalization process. When cells having bound 125I-IL 1 at 4 degrees C were shifted to 37 degrees C, IL 1 moved from the cell membrane to the cytoplasm where it was found in proximity to nuclei or within lysosomes. IL 1 appeared to progressively accumulate in nuclei. Six hours after shifting cells to 37 degrees C, 30 to 35% of the internalized 125I-IL 1 is associated with the cell nucleus. The accumulation of relatively high levels of IL 1 in the nucleus raises the interesting possibility that IL 1 may not only interact in a highly specific manner with cell surface receptors, but also with potentially important nuclear receptors.     

Bacterial internalization mediated by beta 1 chain integrins is determined by ligand affinity and receptor density.

Binding of bacteria to beta 1 chain integrin receptors results in either bacterial adherence or uptake by cultured cells (Isberg, 1991). In this report we show that Staphylococcus aureus coated with high affinity ligands for the beta 1 chain integrin family can be internalized efficiently, whereas bacteria coated with low affinity ligands are poorly internalized. Overproduction of the alpha 5 beta 1 integrin increased the efficiency of bacterial internalization, indicating that the uptake efficiency is directly related to the level of expression of the receptor. By using latex beads or S. aureus coated with mAbs directed against the alpha 5 beta 1 integrin, a roughly semi-logarithmic correlation was observed between the affinity of the receptor-ligand interaction and the rate of bacterial internalization. Evidence is presented that high affinity binding of the bacterium allows the microorganism to compete efficiently with receptor-ligand interactions at the basolateral surface of the cell.     

Identification of IL-1 receptors on human monocytes

The expression and functional analysis of IL-1 beta R on human monocytes were investigated. Binding of 125I-IL-1 to human monocytes was found to be specific and saturable. Scatchard plot analysis revealed a single class of receptors with a binding constant of 600 pM and a receptor density of approximately 100 binding sites per cell. At 37 degrees C 54% of the labeled ligand was internalized over 2 h of incubation. Addition of 0.2% sodium azide to the cells reduced ligand internalization to 9% of total bound. Cross-linking studies revealed that the IL-1R in human monocytes had a Mr of 80 kDa. The addition of IL-1 to monocytes caused changes in membrane Ag expression as assessed by flow cytometric analysis. The results of this study identify IL-1 receptors on monocytes and suggest that IL-1 may act as an effector molecule for monocytes by enhancing expression of Ag correlated with cell differentiation and immune function.     

Upregulation of endothelin 1 and its precursor by IL-1beta, TNF-alpha, and TGF-beta in the PC3 human prostate cancer cell line

Increasing evidence indicates that endothelin 1 (ET-1) is implicated in prostate tumour progression. However, data on ET-1 regulation in human prostate and prostate cancer cell lines are lacking. In this study, regulation of ET-1 and its precursor big ET-1, using PC3 cells, a human bone metastatic prostatic carcinoma cell line, was addressed. ET-1 and big ET-1 assays demonstrated greater secretion of both peptides in the presence of 10% fetal calf serum (FCS) as compared with 0.5% FCS. Incubation of PC3 cells in the absence and presence of various cytokines and growth factors known to be implicated in prostate stroma-epithelium interactions, revealed that IL-6, FGF7/KGF and FGF2/bFGF had no effect on ET-1 and big ET-1 secretion, whereas interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) stimulated their secretion in a concentration-dependent manner. Binding experiments indicated the presence of specific ET-1 receptors in PC3 cells: Kdapp = 1.1 x 0.2 x 10(-10)M, Bmax = 2660 +/- 390 sites/cell. Data analysis demonstrated the presence of only the ETA receptor subtype in PC3 cells. In conclusion, our results indicate that the implication of ET-1 in prostate cancer is likely to be mediated via paracrine/autocrine control of cell factors.     

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

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.