The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors

Cell. Vol. 32, 267-275,

January

1983,

Copyright

0 1983

by MIT

The Asialoglycoprotein Receptor Internalizes and Recycles Independently of the Transferrin and Insulin Receptors Aaron Ciechanover, Alan L. Schwartz*+ and Harvey F. Lodish Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 *and Division of Pediatric Hematology/Oncology Children’s Hospital Medical Center Sidney Farber Cancer institute and Department of Pediatrics Harvard Medical School Boston, Massachusetts 02115

Summary Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. We show that internalization of asialoglycoprotein by HepG2 hepatoma cells is accompanied by a rapid (t,,* = 0.5-l min) depletion of surface asialoglycoprotein receptors. This is followed by a rapid (t,,?: = 2-4 min) reappearance of surface receptors; most of these originate from endocytosed cell-surface receptors. The loss and reappearance of asialoglycoprotein receptors is specific, and depends on prebinding of ligand to its receptor. HepGP cells also contain abundant receptors for both insulin and transferrin. Endocytosis of asialoglycoprotein and its receptor has no effect on the number of surface binding sites for transferrin or insulin. We conclude that binding of asialoglycoprotein to its surface receptor triggers a rapid and specific endocytosis of the receptor-ligand complex, probably due to a clustering in clathrin-coated pits or vesicles. Introduction Receptors for many polypeptide hormones and other protein ligands are distributed diffusely over the cell surface. This has been demonstrated by visualization of fluorescent-labeled ligands such as a2-macroglobulin, insulin, epidermal-growth-factor (EGF) and triiodotyronine in fibroblasts at 4°C (Maxfield et al., 1978, 1979; Schlessinger et al., 1978). In addition, at 4°C ferritin-tagged transferrin was localized diffusely over the erythroid cell surface (Sullivan et al., 1976). Using an immunocytochemical electronmicroscopic approach and antireceptor antibodies, we showed that the asialoglycoprotein receptor is randomly distributed along the entire sinusoidal surface of the rat hepatocyte prior to ligand binding (Geuze et al., 1982). surface ligand-receptor complexes In general, + Present address: Sidney Farber Boston, Massachusetts 02115.

Cancer

Institute,

44 Binney

Street.

([L&J rapidly aggregate into patches upon warming to 37°C. It is not clear that all internalized LR, enter the cell via clathrin-coated pits and vesicles, but many ligands do (for example, asialoglycoprotein, insulin, EGF and low density lipoprotein (LDL); Anderson, et al., 1977a; Maxfield et al., 1978, 1979; Schlessinger et al., 1978; Willingham and Pastan, 1982; Zeitlin and Hubbard, 1982). Endocytosed ligands are then transported to their intracellular destinations via a series of membrane-limited organelles. During this process, ligand-receptor dissociation occurs, probably in a prelysosomal compartment. The ligand is finally found in lysosomes, while the fate of the receptor is not well understood. There is evidence that several receptors recycle to the cell surface. However, the evidence has been largely indirect and the mechanism is not delineated. It has been shown that the ability of cells to internalize ligands far exceeds the total number of binding sites, and that the uptake proceeds in the absence of protein synthesis. Such evidence that surface receptors recycle was obtained for the asialoglycoprotein receptor (Tanabe et al., 1979; Tolleshaug and Berg, 1979; Steer and Ashwell, 1980; Stockert et al., 1980; Schwartz et al., 1982) the mannose-6-phosphate receptor (GonzalezNoriega et al. 1980) the mannose receptor (Stahl et al., 1980) the LDL receptor (Brown et al., 1982) the cup-macroglobulin receptor (Van Leuven et al., 1980) the insulin receptor (Marshall et al., 1981) and the chemotactic peptide receptor (Zigmond et al., 1982). The mechanism that triggers the endocytic cycle is not understood. It is not known whether clustering of receptors into coated pits, internalizing and recycling occurs continuously in the absence or presence of ligand, and if, when it occurs continuously and constitutively, the binding of ligand affects the rate of this process. Earlier data suggest that the LDL receptor recycles even in the absence of ligand. The LDL receptor, however, may be an exception, since most receptors appear to reside in coated pits in the absence of bound ligand (Anderson et al., 1976, 1977b). However, even in the case of the LDL receptor, it was shown that only a portion of the surface receptors cycles continuously, while the other portion can be induced to cycle only by ligand (Basu et al., 1981). Receptors that are randomly distributed on the cell membrane prior to ligand binding may not recycle in the absence of ligand. Kahn et al. (1978) have shown that the binding of insulin to its receptor is a prerequisite to aggregation and internalization of surface receptor complexes. Receptor aggregation has also been demonstrated to be a prerequisite for biological action in other systems, including IgE-mediated mast cell degranulation (Isersky et al., 1978) and EGFstimulated mitogenic activity (Shechter et al., 1979). It is not known whether a specific sorting mechanism exists such that only surface receptors occupied with ligand cluster and internalize, while free surface receptors do not.

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We show that binding of asialoglycoprotein to the asialoglycoprotein receptor of hepatoma cells triggers a rapid internalization of receptor, causing a transient depletion in the number of surface receptors. There is no simultaneous reduction in the number of surface receptors for insulin or transferrin. In addition, we show that replenishment of the surface asialoglycoprotein receptor population also occurs within minutes, and that much of this replenishment is due to endocytosed surface receptors that recycle back to the surface.

A.

Results Internalization of Asialoglycoprotein Is Accompanied by Internalization of Asialoglycoprotein Receptor, Followed by Rapid Return of the Receptor to the Cell Surface Growing HepG2 cells contain 150,000-250,000 functional asialoglycoprotein receptors; 87% of these are on the cell surface and the remaining 13% are internal. Ligand bound at 4°C remains on the cell surface, and is rapidly removed from the cells by treatment with a solution of EDTA. Following warming to 37”C, surface-bound asialoorosomucoid (ASOR) is internalized with a mean half-timeof 2.2 min (Schwartz et al., 1982). The experiment described in Figure 1 B shows that surface asialoglycoprotein receptor, presumably bound to ligand, is also internalized with similar kinetics, and then rapidly returns to the cell surface. Cells were incubated at 4°C for 2 hr in the presence of excess unlabeled ASOR (40 pg/ml; Kd = 0.4 pg/ ml; Schwartz et al., 1981), and thereafter washed free of unbound ligand. The cells were then warmed to 37C in the absence of added ligand for various times ranging from 0.5-l 1 min, and was then quickly rechilled to 4°C (warming and rechilling require approximately 5 set each). Surface receptors unoccupied by ligand were quantified by binding ‘251-ASOR to the cells under saturating conditions. To measure the total number of receptors present on the cell surface, both those occupied and unoccupied with ligand, we first stripped the cells of surface-bound ASOR by incubation for 3 min at 4°C in ice-cold PBS containing 5 mM EDTA. Replicate dishes of cells were then incubated under saturating conditions at 4°C with ‘251-ASOR. After internalization of ASOR, the total number of surface receptors dropped to 45%~55% by 2 min, and then returned to its original value within 8 more minutes (Figure 1 B). Initially, all of the surface receptors were occupied with ligand. All of the receptors that reappeared on the cell surface after one cycle of endocytosis lack bound ligand (Figure 1 B). Following a lag of about 1 min, unoccupied receptors reappear on the surface with a half-time of about 3.5 min. Since control studies (data not shown) demonstrated no loss of prebound ligand to the medium after 15 min incu-

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(A) The calculated curve is shown. The differenhal equation (Schwartz et al., 1982) describing our model of receptor internalization and cycling (see Figure 2) was solved with the following assumptions: At t = 0. (LR), = 0.87, (LR), = 0.13 and R, = 0: k,L = 0 (that is, the absence of free ligand precludes the binding of additional ligand to the cell): k2 = 0.47 min-‘; and k, = 0.23 min-‘. The total number of surface receptors (M) ([LR], + R,) is plotted, normalized to the value of 1.00 at t = 0. Since we assume here that k,L = 0. at the end of the experiment all of the receptors will be on the surface (R, = 1 .OO). and thus there will be slightly more surface receptors than at t = 0. The number of surface receptors free of ligand (R,) is also plotted (M). (B) The experimental curve is shown. HepG2 cells were preincubated for 30 min at 37’C in binding medium containing 0.4 mM cycloheximide and then chilled. They were incubated for an additional 2 hr at 4’C with (M, -1 or without(M) 0.5 gM of unlabeled ASOR in the presence of 0.4 mM cycloheximide. The binding medium was removed and the cells were washed three times in PBS (containing 1.7 mM CaCU at 4°C. The cells were then incubated with 1 ml of prewarmed binding medium (containing 0.4 mM cycloheximide) at 37°C. At the indicated times, the medium was quickly removed and the cells chilled immediately to 0°C by immersion in ice-cold PBS (containing 1.7 mM CaC12). The cells were then treated for 3 min at 4°C in PBS containing 5 mM EDTA C-1 to release the surface-bound ligand. or in PBS alone (M, [1--o). Binding of “?-ASOR at 4°C was performed as described in the Experimental Procedures, except that the medium contained 0.4 mM cycloheximide.

bation at either 4”‘C or 37’C, and since new receptor synthesis was totally abolished by the cycloheximide present throughout the experiment, we feel that all of the measured surface ligand-binding sites at the end of the study originate from those that were originally on the surface and recycled, or from receptors that

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were internal at the start of the study. If no ASOR is prebound to the cell at 4’C (Figure lB), there is no change in the number of surface ASOR receptors subsequent to warming to 37’C. We conclude that the loss and regaining of surface receptors is a consequence of ligand binding and internalization. This experiment directly shows that many surface receptors recycle back to the cell surface. At the start of the 37°C incubation, only 13% of the total population of receptor was internal, while 87% were on the surface (Schwartz et al., 1982). yet 45%-55% of the cell surface receptors disappeared and then reappeared. At least 34% of the receptors on the surface at the end of the study must have been those that were originally on the surface and then internalized and recycled. (At t = 0 and at the end of the study, 0.87 of the total cell receptors were on the surface, and 0.13 were internal. At t = 2 min. 0.44 (= 0.50 x 0.87) were on the surface. Of the surface receptors at the end of the study, 0.74 (= 0.87 - 0.13) must have been on the surface at t = 0. Thus 0.30 (= 0.74 - 0.44) of the total pool of cell receptors were originally on the surface, internalized, and then recycled, or 34% (= 0.30 + 0.87) of those originally on the surface.) Making use of a simple kinetic model for asialoglycoprotein receptor function (Figure 2; adapted from Schwartz et al., 1982) we can calculate a theoretical curve (Figure 1 A) for the number of surface receptors following ligand internalization. We have used the rate constants for internalization of surface receptor-ligand complexes (k,, 0.47 min-‘; mean time of 2.1 min) and for dissociation of internalized receptor-ligand complexes and return of the receptor to the surface (k,, 0.23 min-‘, mean time = 4.2 min), which we calculated previously (Schwartz et al., 1982). We regard the agreement of experimental results in Figure 1B with our prediction as marked confirmation both of our model and of the calculated values of ka and k,. We conclude that surface asialoglycoprotein receptor is internalized in parallel with ligand, with a first-order rate constant of about 0.47 min-‘, and that receptor returns to the surface with a rate constant of about 0.23 min-’ (the experimental value is 0.32 min-‘, taken from Figure 1 B). Binding of ASOR to the cell surface induces internalization of the asialoglycoprotein receptor. Does binding of ASOR cause a general redistribution of surface proteins? In particular, does it cause internalization of other surface receptors? Before answering this question, we need to show that HepG2 cells contain functional receptors for other ligands (that is, transferrin and insulin). Transferrin Receptors on HepGP Cells The number of transferrin receptors on HepG2 cells were determined and their affinity for “51-transferrin

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(L) ligand; [(R),] unoccupied surface receptors; [(LR),] occupied surface receptors; [(LR),] occupied internal receptors; [(RI] unoccupied internal receptors; (k,) rate constant for binding; (k2) rate constant for internalization; (k3) rate constant for dissociation of ligand and receptor within the cell; (k,) rate constant for reappearance of receptor to cell surface; (k,) overall rate constant for dissociation of receptor-ligand complex and return of internal receptor to cell surface (1 /k, = 1 /k, + 1 /k,).

by analysis of the concentration-dependent binding (Figure 3). The cells were incubated at 4°C a temperature at which only surface receptor binding occurs. There are approximately 5.1 X lo4 receptors per cell surface (mean of three experiments; range 4.6-6.0 X :04). The apparent dissociation constant calculated from a Scatchard (1948) analysis (Figure 3) of these data was Kd = 4.4 X lo-’ M. Unlabeled transferrin competed equally with lz51-transferrin for cell surface binding sites in simultaneous competition assays (data not shown). This demonstrates retention of full biological activity of the ‘251-labeled transferrin. Binding of ‘z51-transferrin was performed at 4°C in the presence of either 1 mg/ml of bovine serum albumin, ovalbumin, myoglobin, hemoglobin or cytochrome c, or 250 pg/ml of lactoferrin, ferritin or ASOR. Although bovine serum albumin and hemoglobin inhibited the I25 I-transferrin binding, probably nonspecifically, by approximately 20% (data not shown), none of the other proteins examined demonstrated any competition for ‘251-transferrin binding. To determine the extent of transferrin internalization by HepG2 cells, we used the procedure of Karin and Mintz (1981) to discriminate between surface-bound and internalized ligand. ‘?-transferrin was bound at 4°C to HepGP cells as described in the Experimental Procedures. Unbound ligand was removed by wash-

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Binding was measured at the indicated concentrations described in the Experimental Procedures. (M) total binding: (A-A) nonspecific binding; (M) specific binding. Insert: Scatchard analysis of the binding data. Ordinate: ml/mg protein. Abscissa: fmoles/mg protein.

ing, after which the cells were incubated for various times at either 4°C or 37°C. Thereafter, all cells were treated with pronase at 4°C for 1 hr. Only surfacebound ligand is accessible to the proteolytic enzyme and is released into the medium, whereas the internalized ligand is protected from proteolysis and recovered with the cell pellet. In the experiment shown in Figure 4, 95% of the ‘*?-transferrin that was bound to the cells at 4°C was liberated by pronase. Warming the cells to 37°C prior to the pronase treatment for only 3 min rendered about 50% of the cell-associated ‘251-transferrin pronase-resistant. After 6 min at 37°C the pronase-resistant fraction reached its maximum value, 70% of the cell-associated ligand. Additional experiments (A. Ciechanover, A. Dautry-Varsat, H. F. Lodish, manuscript in preparation) have demonstrated that internalized ‘251-transferrin diacytoses through the HepG2 cell rather than being degraded by the lysosomes, consistent with the observations of others about other cells (Kailis and Morgan, 1974; Hemmaplardh and Morgan, 1976; Karin and Mintz, 1981; Octave et al., 1981; Bridges et al., 1982a). Insulin Receptors on HepG2 Cells Results of concentration-dependent binding of ‘*?insulin to HepG2 cells at 4°C are seen in Figure 5. Replotting these data via Scatchard (1948) analysis

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Binding was performed as described in the Experimental Procedures. At the end of the incubation, the cells were washed three times in icecold PBS (containing 1.7 mM CaC12; zero time values were 353 and 21 fmoles/mg protein for total and nonspecific binding, respectively). The cells were then incubated with 1 ml of binding media containing 100 nM unlabeled transferrin at either 4’C (M) or 37°C (M) prewarmed to the appropriate temperature. At the indicated times, the binding buffer was removed, and the cells were treated with pronase as described in the Experimental Procedures. The percent of cell-associated ‘%transferrin that was pronase-resistant is plotted against the second incubation time. Data have been corrected for nonspecific binding.

yields a curvilinear relationship (Figure 5), suggestive of either heterogeneity of sites (Kahn et al., 1974; Pollet et al., 1977) or negative cooperativity (DeMeyts et al., 1973, 1976). We found a negligible effect of excess free unlabeled insulin on the dissociation at 4°C of surface-bound ‘251-insulin (data not shown). These data are consistent with two independent components of high and low affinity. From the Scatchard analysis (Figure 5), we estimate 3 X 1 O4 high-affinity binding sites per cell surface with apparent Kd of approximately 3 X 1 O-’ M. In addition, there may be as many as 7.5 x 1 O4 low-affinity sites per cell surface with apparent Kd of approximately 6 x 1 O-’ M. We examined the binding of various ratios of ‘251labeled and unlabeled insulin at 100 nM total insulin concentration. The cell-associated radioactivity was always z 90%, that expected from the dilution of the ‘251-insulin (data not shown), indicating that essentially all of the ‘251-labeled insulin molecules are biologically active. Furthermore, we have shown via SDS-polyacrylamide gel electrophoresis and autoradiography that the bound ligand is indeed the ‘251-labeled insulin (or transferrin) rather than other possible contaminants in the preparation (data not shown). Binding of ‘*‘l-insulin was not altered by the presence of bovine serum albumin, ovalbumin, bacitracin or cytochrome c at concentrations of 1 mg/ml (data not shown). These

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Binding was determined at the indicated concentrations as described in the Experimental Procedures. (U) Total binding; (A-A) nonspecific binding; (M) specific binding. Insert: Scatchard plot analysis of the binding data. Ordinate: ml/mg protein. Abscissa: fmoles/mg protein.

data confirm the specificity of the ‘z51-insulin binding sites on HepG2 cells. It should be noted that the binding of both insulin and transferrin at 4°C is timedependent, with saturation of most surface sites reached after approximately 2 hr (data not shown). The internalization of surface-bound insulin was studied with a different approach from that used for transferrin; the entire incubation at either 4’C or 37°C was performed in the presence of ‘251-insulin in the medium, since insulin prebound to the cells at 4°C is rapidly released to the medium upon warming, without being internalized. As can be seen in Figure 6, 15% of the cell-associated ‘251-insulin becomes pronaseresistant after 2 min at 37’C, compared with 6% in cells maintained at 4°C. By 6 min at 37°C 28% is protected from the proteolytic enzyme. Thus the data in Figures 4 and 6 show that both transferrin and insulin are very rapidly and progressively internalized by the hepatoma cells at 37°C with half-lives of approximately 2-4 min. Are the Receptors for Asialoglycoproteins, Insulin and Transferrin Internalized Independently? To investigate whether receptor-mediated endocytosis of one ligand is accompanied by simultaneous depletion of the binding sites of other ligands, we made use of the experimental protocol described in the legend to Figure 1 B. Binding of ASOR causes a rapid reduction in the number of surface asialoglycoprotein receptors. However, there is no alteration in

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HepG2 cells were incubated with 100 nM ‘251-insulin at either 4°C (M) or 37°C (U). At the indicated times, the binding buffer was removed and the cells were washed three times in ice-cold PBS (containing 1.7 mM CaC12) and were treated with pronase as described in the Experimental Procedures. The percent of cell-associated ‘251-insulin that was pronase-resistant is plotted against incubation time. Data have been corrected for nonspecific binding.

the number of surface binding sites for either transferrin (Figure 7A) or insulin (Figure 78). We conclude that binding of ASOR induces a highly specific internalization only of its own receptor. Discussion Our principal goal was to determine the dynamics of one cell-surface receptor-that for asialoglycoproteins-subsequent to ligand binding. We also wanted to determine whether the endocytosis of one ligand-receptor complex is associated with the internalization of the surface receptors of an independent ligand. Toward these goals, we identified and characterized three independent polypeptide receptors on the human hepatoma cell HepG2. Among the advantages of this monolayer cell line in studies of receptor function is the possibility of performing certain rapid manipulations that are difficult to perform in suspension or primary cultures of hepatocytes. These manipulations were crucial for the performance of the key experiments presented (for example, less than 5 set required to chill cells from 37°C to 4°C). In addition, this cell line presents a metabolically stable population of defined cells that is not altered from preparation to preparation as are fresh hepatocytes. We have already shown that these cells express substantial abundance of the asialoglycoprotein receptor that binds and internalizes ‘“?-ASOR

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Figure 7. Independent Internalization protein, Insulin and Transferrin

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Cells were treated as described in the legend to Figure 1B. After “stripping” of the noninternalized ASOR with EDTA, the binding of labeled ASOR. insulin and transferrin was measured as described in the Experimental Procedures, except that the insulin concentration was 200 nM. (A) Zero time values for ASOR were 1147 and 209 fmoles/mg protein for total and nonspecific binding, respectively, and 391 and 47 for transferrin. (B) Values were 1096 and 21 for ASOR and 374 and 69 for insulin, respectively. (A) ASOR: U. Transferrin: M. (B) ASOR: W. Insulin: H. All data shown represent averages of quadruplicate determinations and were corrected for nonspecific binding.

(Schwartz et al., 1981, 1982). Here we showed that these cells also express functional receptors for transferrin and insulin. There are approximately 50,000 high-affinity transferrin binding sites per cell surface with an apparent Kd or 4.4 X lo-’ M. We have interpreted the curvilinear Scatchard analysis (Figure 5) to suggest the presence of two populations of insulin receptors; approximately 30,000 high-affinity sites per cell surface and at least 70,000 low-affinity sites per cell surface with apparent Kd of 3 X 1 O-’ M and 6 x IO-* M, respectively. However, these data are also consistent with one population of receptors

that exhibit negative cooperativity (see Results). It should be noted that the insulin binding assays were carried out on cells in monolayer and at 4”C, different from the routine assays of insulin binding to cells (that is, in suspension at 18°C; Rubin et al., 1978; Grunfeld et al., 1980). This may contribute partially to the curvilinear character of the binding curve and to the nonsaturability of the receptors, although we believe these differences are minor. Once bound, both transferrin and insulin are rapidly internalized at 37°C. This is seen by the rapid acquisition of resistance to extracellular pronase. Within 3 min, 50% of the cell-associated transferrin molecules were internalized (Figure 4). We could not use the same experimental approach for measuring insulin internalization, since upon warming the cells to 37°C prebound insulin rapidly dissociates from its surface receptor in the absence of internalization (data not shown). However, when ‘251-insulin is presented to cells for 2 min at 37°C 15% of the cell-associated insulin becomes resistant to extracellular pronase (Figure 6). Although in this case we cannot determine the precise mean time required for the internalization of (LR), complex, we can conclude that this process occurs very rapidly. The mean time required for internalization of asialoglycoprotein bound to the cell surface is 2.2 min (Schwartz et al., 1982). If the internalization of a surface receptor for a particular ligand is a specific process, and if all surface receptors are internalized in synchrony, one should be able to detect a significant depletion of surface receptors for a short period of time, until the depleted pool is replaced. Even if the process of receptor internalization occurs continuously both in the presence and absence of bound ligand (thus making our assumption incorrect), it is likely that accelerated internalization or decreased externalization occurs in the presence of ligand, causing depletion of surface receptors. This assumption was based on our previous finding that in the continuous presence of ASOR, there is a substantial redistribution of functional asialoglycoprotein receptors to intracellular sites. The number of functional intracellular receptors is doubled (13%-28% of total) when HepG2 cells are continuously exposed to 2 pg ASOR/ml (Schwartz et al., 1982). Indeed, we were able to demonstrate a 55% reduction in cell-surface asialoglycoprotein binding sites after saturating the cell-surface sites with ASOR at 4°C and then warming the cells to 37°C (Figures 16 and 7). This observation is consistent with our calculations based on the functional receptor distribution and the kinetics of ASOR receptor internalization and recycling as discussed above (Figure 1A; Schwartz et al., 1982). The inability of others (Wall and Hubbard, 1981; Bridges et al., 1982b) to demonstrate such a reduction in surface receptor sites subsequent to ASOR binding is probably due to the inability to manipulate cells in suspension or organ

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perfusion at short intervals, and to the particular distribution of the binding sites in this cell between the cell surface and the intracellular compartment(s). While in HepG2 cells the majority (87%) of the functional receptors are on the cell surface (Schwartz et al. 1982) in isolated rat hepatocytes the majority of the binding sites appear to be located intracellularly (Pricer and Ashwell, 1976; Steer and Ashwell, 1980). The fraction of the hepatocyte asialoglycoprotein receptors that is on the cell surface is controversial (Schwartz et al., 1980). It might well be that in the hepatocyte, but not in HepGP cells, internalized receptors are replaced immediately by newly recruited receptors coming from an intracellular pool. Despite a transient 55% reduction in ASOR surface receptor sites, there was no reduction in the surface binding capacity for either transferrin (Figure 7A) or insulin (Figure 76). This suggests that the transferrin and insulin receptors are not internalized under these conditions, or that their rate of internalization is not changed (either by an increase of the rate of internalization or by a decrease in the rate of their externalization) during the uptake of other ligands via receptormediated endocytosis. We were unable to perform the reciprocal experiments of internalizing either transferrin or insulin and measuring the binding capacity of ASOR, since neither of these ligands can be released from their receptors in a way that allows additional binding assays. We conclude that the process of asialoglycoprotein receptor internalization is at least partially (if not completely) specific, and is dependent upon the preoccupation of the surface receptor by its appropriate ligand. The mechanism of surface receptor sorting prior to internalization is not clear. It may involve changes in the rate of lateral movement of the surface receptor after binding of the ligand. Lateral diffusion alone cannot account for the thermal sensitivity of patch formation and endocytosis (Hillman and Schlessinger, 1982) suggesting the participation of specific mechanoskeletal factors (Hemmaplardh et al., 1974). Further studies directed toward the structure of cell surface receptors and their topology in the membrane will help to shed light on this question. The rapid depletion of the surface ASOR receptors was followed by a similarly rapid replacement at the cell surface (Figures 1B and 7). The use of cycloheximide, and the fact that the binding and the uptake rate of ASOR do not change in the presence of this protein synthesis inhibitor (Schwartz et al., 1982). show that the newly appearing surface receptors do not depend upon de novo synthesis of new molecules. The question arises as to the source of the renewed surface receptors. Are they previously internalized and recycled, or are they from an intracellular pool, previously unavailable to ligand? We have demonstrated in HepGP cells that only a minor portion of the

functional ASOR receptors are intracellular (-13%), while the vast majority of functional receptors are on the cell surface (Schwartz et al., 1982). Since the binding capacity is decreased to as low as 45% of the initial value after internalization of the ligand-occupied receptors, we suggest that the renewed binding sites are recycled surface receptor molecules, rather than molecules from a large intracellular pool (work in progress). There is an alternative explanation for our results. It is possible that internalization of asialoglycoprotein receptor occurs at the same rate (tYj2 = l-2 min) whether or not ligand is bound. The recycling of receptor to the surface could be slowed dramatically if ligand is bound to it, perhaps because a different pathway is used. It should be noted, however, that we were unable to see depletion of cell-surface receptors even after very short times of warming (0.5 min) without prebinding of a ligand. In contrast to our findings, a portion of the LDL receptor appears to cycle independently of ligand binding (Basu et al., 1981); perhaps the pathway for recycling of LDL receptor with or without bound ligand is the same. Internalized receptors reappear on the cell surface very quickly, with a mean time of about 3-5 min (Figure 1 B). Studies in isolated hepatocytes and in intact liver showed that internalized asialoglycoproteins appear in lysosomes, but only after 1 O-20 min. Moreover, immunoelectronmicroscopic studies demonstrated ASOR receptor in intracellular vesicles just under the cell surface, but not in lysosomes (Geuze et al., 1982). Together with our kinetic studies, these data suggest that the receptor loses its ligand in an intracellular prelysosomal compartment and that the receptor itself never reaches the lysosome. Experimental

Procedures

Cells All experiments HepGP (Knowles conditions were

were carved out with the human hepatoma Ime. et al., 1980). Maintenance of the cells and growth performed as described by Schwartz et al. (1981).

Materials Iron-free human transferrin was purchased from Calblochem. Orosomucoid was purchased from the American Red Cross, and crystalline pork zinc insulin was gift from J. Schlichtkrul. Eagle’s minimal essential medium (MEM) and fetal calf serum were purchased from Gibco. Na-‘? was obtained from Amersham. All other chemicals were reagent grade.

lodination

of Proteins

All proteins were labeled by a slightly modified chloramine-T method (Greenwood et al., 1963). ASOR was prepared from orosomucoid and iodinated as described by Schwartz et al. (1980). Transferrin was saturated with Iron and iodinated as described by Karin and Mintz (1981). The specific activity was generally about 4 X 1 O8 cpm/nmole of protein. Insulin was iodinated for 4 min essentially as described by Freychet et al. (1971) and as modified by Starr and Rubenstein (1979). The specific activity was approximately 1 .7 X lo9 cpm/ nmole of protein. All labeled ligands were used withm 3 weeks of preparation.

Cdl 274

Binding Assays All binding assays were carried out in tissue-culture dishes (35 mm; Falcon) which were generally seeded 4 days prior to assay (0.6-l .O x lo6 cells/ per dish). At the time of assay, the dishes were near confluence (2.0-3.0 x lo6 cells per dish). The cells were washed twice at 4°C with MEM. buffered with 20 mM of 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid (pH 7.3), and gassed with 95% O2 and 5% COP (binding medium). They were preincubated for 20 min at 37°C in binding medium. Unless otherwise stated, binding was carried out in duplicate dishes at 4°C for 2 hr in binding medium with 100 nM each of labeled ASOR. insulin and transferrin. Nonspecific binding was determined in the presence of a 1 OC-fold excess of the appropriate unlabeled ligand. After washing off unbound ligand, the ligand bound to cells was measured (Schwartz et al., 1981). Pronase Treatment of Cells Cells were treated with pronase as described by Karin and Mintz (1981), except that for the determination of insulin internalization the cells were incubated with pronase for 2.5 hr. Briefly, after washing off excess unbound labeled insulin or transferrin, each plate was incubated with 1 ml of 0.25% pronase in binding buffer at 4’C. At the end of the incubation, the cells were completely detached from the dish by repeated pipetting, and were centrifuged for 1 min in an Eppendorf microfuge. The supernatant was separated from the pellet and the radioactivity of each fraction was determined.

This study was supported by grants from the National Institutes of Health. A. C. is supported by the Melvin Brown Memorial Fellowship through the Israel Cancer Research Fund, and by a postdoctoral fellowship from the Leukemia Society of America. A. L. S. is supported in part by The Medical Foundation Inc. We thank Miriam Boucher for her skillful help in the preparation of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. October

9. 1982

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