Binding, internalization, and intracellular processing of protein ligands. Derivation of rate constants by computer modeling

Information about ligand binding, dissociation, internalization, and intracellular processing and about receptor turnover, processing, and insertion into the membrane is contained in the time-dependent changes in concentrations of membrane-associated and internalized ligand. Single experiments similar in design to those typically performed for Scatchard analyses of binding data conducted at physiological temperature and in the absence of inhibitors of ligand-receptor complex internalization and degradation can provide kinetic data sufficient to permit derivation of all the respective rate constants by numerical methods. We developed an analytical solution of the kinetic model which assumes that all of these processes follow first order kinetics. The model represents interactions of surface receptors (R)s, the surface ligand-receptor complex (LR)s and internalized receptor-ligand complex (LR)I: d[R]S/dt = Vr - kt[R]S - ka[L] [R]S + kd [LR]S; d[LR]S/dt = ka[L] [R]S - kd[LR]S - ke[LR]S; d[LR]I/dt = ke[LR]S - kh[LR]I; Vr is the constant rate of insertion of receptors into the membrane, kt is the internalization rate constant for free receptors, ka and kd are association and dissociation rate constants for ligand-surface receptor interaction, ke is the internalization rate constant for ligand-receptor complexes, and kh is the intracellular ligand decomposition rate constant. The interaction of radioiodinated human recombinant interferon-alpha 2a with the human alveolar lung carcinoma cell line, A549, was adequately accounted for by the model. The rate constants, numerically derived from time-dependent concentrations of surface-bound and internalized ligand of other systems taken from the literature, were in agreement with values of these rate constants individually measured by steady-state experiments. In cases where the fate of internalized radioactivity was more complex than assumed by the model, the parameters ka, kt, (kd + ke) and Vr could be derived from the time dependence of [LR]S.     

Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism.

The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21-32, 1989). To determine whether E5 affects EGF:receptor interactions we analyzed the kinetics of 125I-EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I-EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy-independent in both cell lines. The 125I-EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy-dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I-EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy-independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor.     

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.     

The effects of chronic ethanol administration on the rates of internalization of various ligands during hepatic endocytosis.

The purpose of the present study was to further characterize the ethanol-induced impairments in hepatic endocytosis. Specifically, we examined the effects of ethanol treatment on receptor-ligand internalization via the coated and noncoated pit pathways. Insulin, epidermal growth factor (EGF) and asialoorosomucoid (ASOR) were used as model ligands to study internalization by isolated hepatocytes. ASOR and EGF are thought to be internalized strictly in coated pit regions of the cell membrane, while insulin may be internalized in both coated and uncoated membrane regions. Ethanol administration for 5-7 weeks decreased internalization of ASOR and EGF while internalization of insulin was unchanged during a single round of endocytosis of surface-bound ligand. Similarly, a more quantitative measure of endocytosis, the endocytic rate constant, was decreased for EGF and ASOR but not for insulin in livers of experimental rats. When endocytosis of Lucifer yellow, a fluorescent dye known to be internalized in the cell by fluid-phase endocytosis was examined, the initial rates of dye uptake were not significantly altered by alcohol administration. These results indicate that ethanol may selectively impair internalization occurring by coated pits while it has a minimal effect on initial uptake of molecules which are internalized by noncoated membrane regions.     

Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics

We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.     

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227]

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.     

Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.     

Analysis of the steady state binding, internalization, and degradation of human interferon-alpha2

Scatchard analyses of the equilibrium binding of radiolabeled human interferon-alpha2 (huIFN-alpha2) to Madin-Darby bovine kidney cells previously exposed to subsaturating concentrations of IFN-alpha showed approximately a 50% decrease in the number of cell surface receptors and no change in the apparent dissociation constant, Kd, compared with cells not exposed to interferon. The steady state equations describing the interaction of polypeptide ligands with cell surface receptors under physiological conditions (Wiley, H.S., and Cunningham, D.D. (1981) Cell 25, 433-440) have allowed us to determine, under steady state conditions, the rate of insertion of receptors into the cell membrane, the endocytic rate constant of occupied receptors, the rate constant of turnover of unoccupied receptors, and the rate of hydrolysis of internalized ligand. Our results indicate that occupied and unoccupied interferon receptors are cleared from the cell surface at approximately the same rate. This suggests that the down-regulation of the huIFN-alpha2 receptor on Madin-Darby bovine kidney cells by huIFN-alpha2 differs from that of several other surface receptors for polypeptide hormones and growth factors analyzed on cultured cells in that the binding of huIFN-alpha2 to its receptor does not increase the rate of receptor endocytosis.     

The fate of IgE bound to rat basophilic leukemia cells. II. Endocytosis of IgE oligomers and effect on receptor turnover

We have assessed the internalization of variously sized oligomers of IgE bound to rat basophilic leukemia (RBL) cells by measuring their accessibility to the extracellular environment, and by direct visualization of the radiolabeled ligands. We also followed the fate of the internalized ligands and their receptors, as well as the fate of the free receptor on cells internalizing oligomers. In contrast to monomeric IgE, surface-bound oligomeric IgE was internalized. Notably, dimers provided an effective signal for internalization, although larger oligomers seem to be internalized more efficiently. In our experiments, 48% of the cell-bound dimers and 67% of the trimers were eliminated from the cell surface in 180 min. One-half of the maximal internalization observed with dimers and trimers occurred in 25 and 11 min, respectively. Release of radioactivity into the supernatant followed internalization; the released radioactivity did not bind to fresh cells and was only partially TCA-precipitable. Radioactive ligands remaining associated with the cells were unchanged as judged by m.w; they also were shown to remain receptor-bound. During either internalization or release of substantial amounts of the originally cell-bound oligomers, there was no increase in IgE-binding activity. In contrast, there was a transient drop (25%) in the number of free surface receptors suggesting internalization of the free receptors together with the oligomer-occupied receptor. Cells that failed to release histamine (RBL-I) processed dimeric and trimeric IgE similarly to histamine-releasing (RBL-2H3) cells. We conclude that dimeric and trimeric IgE are internalized by RBL cells and later are released to the medium in a partially degraded form. The ligand-bound receptor seems to be internalized with the ligand, along with some free receptor, and does not appear to be reusable or to recycle rapidly to the cell surface.     

Quantitative studies of the rate of insulin internalization in isolated rat hepatocytes.

We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.     

Dynamics of interleukin-6 internalization and degradation in rat hepatocytes.

We have investigated the dissociation, internalization, and degradation of 125I-interleukin-6 (125I-IL-6) by primary rat hepatocytes. Temperature shift experiments following saturation binding of 125I-IL-6 to cell surface receptors in hepatocytes showed a rapid loss of surface-bound 125I-IL-6 (t1/2 = 15 min), concomitant with a rapid rise in internalized radiolabeled ligand. After reaching a maximum by 30 min at 37 degrees C, the level of internalized 125I-IL-6 decreased with time and appeared in the culture media in a non-trichloroacetic acid-precipitable (degraded) state. The addition of the lysosomotropic agent chloroquine inhibited this receptor-mediated degradation of IL-6 without affecting ligand internalization. Polyacrylamide gel electrophoresis analysis of internalized 125I-IL-6 confirms these results. Additionally, we show that the IL-6.IL-6 receptor complex is stable, and dissociation of these two molecular species occurs at a pH below 5.0. In contrast to published results, data presented in this study clearly indicate that IL-6 is rapidly internalized and degraded within hepatocytes by a receptor-mediated mechanism.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments

We have examined the rate of dissociation of internalized 125I-asialo-orosomucoid-receptor complexes in freshly isolated rat hepatocytes. Cell suspensions were washed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 0 degrees C to remove surface-bound ligand and then assessed for the retention of radioactive glycoprotein in the presence of digitonin, which permeabilized the cells and released the internal soluble contents. In cells which initially contained only surface-bound ligand, about 50% of the internalized ligand dissociated from receptor very rapidly (t1/2 less than or equal to 2.5 min, k greater than or equal to 0.28 min-1), at 37 degrees C, whereas the other 50% dissociated more slowly with apparent first order kinetics (t1/2 = 50 min, k = 0.014 min-1). This equal distribution of internalized ligand into two compartments, from which dissociation occurred with very different kinetics, did not depend on the extent of surface receptor occupancy and also occurred under non-steady state conditions of continuous exposure to ligand. Ligand entering both the rapid and slow dissociation compartments was eventually degraded with apparent first order kinetics (k = 0.0047 min-1), suggesting that the intracellular routing of ligand to lysosomes after dissociation from either compartment was via the same pathway. The fast and slow dissociation of receptor-ligand complexes were also distinguished by different temperature sensitivities; the slow dissociation process ceased below 18 degrees C, whereas the fast dissociation process still proceeded. The equal partition of internalized complexes into the two kinetic compartments did not change as a function of temperature but did change as cells continued to endocytose asialo-orosomucoid at 37 degrees C. As the internal receptor pool approached a steady state level of occupancy, there was an increase in the average time for receptor recycling and an increase in the fraction of incoming receptor-ligand complexes which dissociated rapidly (approximately 75%). In addition, under steady state conditions, the rate of the slow dissociation process increased (k = 0.026 min-1, t1/2 = 27 min).     

Reaction rate enhancement by surface diffusion of adsorbates.

Ligands can be captured by a surface target through either direct bulk diffusion or surface diffusion following reversible adsorption to the surface. We have solved a steady state boundary value problem for a perfect sink disk target in the surface, taking into account bulk and surface diffusion coefficients D and Ds and adsorption/desorption kinetic rate constants ka and kd at non-target regions. Solutions have been successfully found by numerical computation. The results show that the rate of capture from the surface depends non-linearly on Ds, D, ka, kd and geometrical dimensions. In particular, we demonstrate that not only is the non-target region equilibrium constant Keq (= ka/kd) important in determining the rate of capture from the surface, but so are the kinetic rate constants ka and kd separately. In all cases, the surface adsorption/diffusion combination enhances the total rate of capture. The results should be useful for predicting reaction rates of biological membrane bound receptor clusters and substrate-immobilized enzymes.     

The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine

In this study, we characterized and compared the ligand-independent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes treated with monensin, chloroquine, microtubule depolymerizing agents, or NaN3 and NaF at 37 degrees C. Freshly isolated hepatocytes exhibit predominately one subset of surface Gal receptors, termed State 1 receptors (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). During equilibration at 37 degrees C, these cells also express a second subset of Gal receptors at the surface, termed State 2 receptors, and routinely double their total surface Gal receptor activity. Following equilibration at 37 degrees C and then inhibitor treatment, hepatocytes bound 40-60% less 125I-asialoorosomucoid (ASOR) at 4 degrees C than did untreated cells. Treated cells maintained a basal nonmodulated level of surface receptor activity regardless of temperature, perturbant concentration, or incubation time. Loss of surface Gal receptor activity on cells treated with multiple inhibitors simultaneously or sequentially was not additive. Thus, all treatments affected the same subpopulation of surface Gal receptors. None of these inhibitors decreased surface State 1 Gal receptor activity, but all prevented the normal appearance of State 2 Gal receptors on freshly isolated cells during incubation at 37 degrees C. The endocytic capability of residual surface State 1 Gal receptors on inhibitor-treated cells varied depending on the inhibitor. Hepatocytes treated first at 24 degrees C or with colchicine at 37 degrees C internalized greater than 85% of surface-bound 125I-ASOR. In contrast, monensin- or chloroquine-treated cells internalized approximately 50% of surface-bound 125I-ASOR. Azide-treated cells internalized less than 20% of surface-bound 125I-ASOR. We conclude that only surface State 2 Gal receptor activity is sensitive to these various perturbants. State 1 Gal receptor activity is not modulated. These data are consistent with the conclusion that only State 2 Gal receptors constitutively recycle.     

rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.     

Chronic ethanol administration impairs the binding and endocytosis of asialo-orosomucoid in isolated hepatocytes

We have examined the effect of ethanol administration on receptor-mediated endocytosis of asialo-orosomucoid by isolated hepatocytes. Significantly less ligand was bound, internalized, and degraded by hepatocytes isolated from rats fed an ethanol diet for 5-7 weeks than by cells isolated from chow-fed or pair-fed controls. Reduced binding was shown to be primarily due to a decreased number of cell surface receptors rather than to a lowered affinity of the receptor for its ligand. This reduction in cell surface receptors resulted in a marked inhibition of internalization and degradation of ligand by hepatocytes from the ethanol-fed rats. In addition, a defect in the initial stages of receptor-ligand internalization was also indicated, since less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared to controls. Rates of internalization and degradation of internalized ligand were, however, similar for all three groups, suggesting that neither degradation per se nor rate of delivery of internalized ligand to the lysosomes was affected by ethanol feeding. Receptor recycling was impaired in ethanol-fed rats, as indicated by a decrease in the binding site number after stimulation of endocytosis for 120 min when compared to initial binding capacity. Receptor recycling was not impaired in hepatocytes from control animals. These results indicate that chronic ethanol feeding impairs the process of receptor-mediated endocytosis by the liver; the major cause of this impairment appears to be due to a decreased number of cell surface asialoglycoprotein receptors in the ethanol-fed animals, along with a decreased ability of these cells to internalize all of the surface-bound ligand.     

Kinetics of insulin internalization and processing in adipocytes: effects of insulin concentration

We have studied the effect of insulin concentration on the kinetics of insulin internalization and efflux in isolated rat adipocytes. To determine internalization rates adipocytes were incubated with 125I-insulin at 37 degrees C; and at frequent, early time points surface-bound and intracellular insulin were quantitated. Surface-bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3.0 buffer at 4 degrees C. From this data the endocytotic (internalization) rate constant (ke) was calculated for six insulin concentrations ranging from 0.3 to 100 ng/ml. Ke was found to decrease in an insulin concentration-dependent manner (P less than .001). Thus, values for ke were 0.121 +/- 0.006 min-1 versus 0.074 +/- 0.011 min-1 at 0.3 ng/ml and 100 ng/ml, respectively. The decrease in ke did not parallel insulin concentration-dependent changes in insulin receptor affinity indicating it was not the result of an inability of low affinity receptors to be internalized. The kinetics of insulin efflux were determined by loading various concentrations of 125I-insulin into the adipocyte interior, washing away surface-bound and extracellular insulin, and then monitoring the subsequent efflux of pre-loaded insulin into medium that contained the same concentration of insulin used in the loading step. The overall rate of efflux was independent of insulin concentration. In summary, these results show that at high insulin concentrations the efficiency of insulin internalization is impaired. In contrast, the rate of insulin efflux is unaffected.     

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.     

Rapid constitutive internalization and externalization of epidermal growth factor receptors in isolated rat hepatocytes. Monensin inhibits receptor externalization and reduces the capacity for continued endocytosis of epidermal growth factor

It was previously demonstrated that freshly isolated rat hepatocytes can internalize severalfold more epidermal growth factor (EGF) molecules than the number of surface EGF receptors, suggesting extensive reutilization of receptors during endocytosis (Gladhaug, I. P. & Christoffersen, T. (1987) Eur. J. Biochem. 164, 267-275). The present report attempts to explore the pathways involved in the externalization of EGF receptors. Incubation of hepatocytes at 37 degrees C in the absence of ligand increased the surface receptor pool by 50-100% within 45 min. Pretreatment with monensin inhibited the turnover of the surface EGF receptor pool by 50-60% within 10 min and blocked the temperature-dependent externalization of receptors. Cycloheximide caused a slower attenuation of the surface receptor pool, whereas tunicamycin and chloroquine did not significantly affect the exchange of receptor pools. Monensin reduced the surface receptor pool and the endocytic uptake in corresponding proportions, without affecting the internalization of prebound EGF. Endocytic uptake was unaffected by chloroquine and slightly reduced by cycloheximide. The internalization of unoccupied receptors and the endocytosis of prebound EGF followed similar kinetics (t1/2 approximately 5 min), suggesting that unoccupied receptors are internalized at a rate comparable to that of occupied receptors. The results suggest that there is a rapid turnover of the surface pool of EGF receptors with constitutive internalization of unoccupied surface receptors and externalization of internal receptors. This is consistent with, but does not prove, a true recycling of the EGF receptors in the hepatocytes. The monensin-sensitive externalization pathway determines the capacity for continued endocytosis of EGF.     

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.