©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Trafficking of Epidermal Growth Factor Family Ligands Is Directly Influenced by the pH Sensitivity of the Receptor/Ligand Interaction (*)

(Received for publication, September 13, 1994; and in revised form, December 1, 1994)

Anthony R. French (1) Douglas K. Tadaki (3) Salil K. Niyogi (3) Douglas A. Lauffenburger (1) (2)

From the  (1)Departments of Chemical Engineering and (2)Cell & Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 and the (3)Protein Engineering and Molecular Mutagenesis Program and University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using members of the epidermal growth factor (EGF) family as well as site-directed recombinant human EGF mutants, we investigated how ligand binding properties influence endosomal sorting. Mouse EGF (mEGF), human EGF (hEGF), and transforming growth factor alpha (TGFalpha) bind to the human EGF receptor (EGFR) with similar affinities at pH 7.4. However, the binding properties of these ligands have substantially different pH sensitivities resulting in varying degrees of dissociation from the receptors at lower pH levels characteristic of endosomes. We employed a steady-state sorting assay to determine the fraction of ligand sorted to recycling versus degradation as a function of the number of intracellular ligand molecules in mouse B82 fibroblasts. mEGF, hEGF, and TGFalpha display significantly different steady-state endosomal sorting patterns which correspond to the extent of their dissociation at endosomal pH. Moreover, several recombinant hEGF mutants with differing affinities exhibit altered endosomal sorting compared to hEGF, demonstrating a similar direct relationship between ligand binding properties and endosomal sorting outcomes. Intracellular trafficking of the EGF ligands was also monitored by measuring the observed degradation rate constants. These likewise show marked differences that correlate with the differing pH sensitivities of the ligands' binding properties.


INTRODUCTION

Eukaryotic cells are able to direct and maintain substantial flows of intracellular protein and membrane traffic. Newly synthesized proteins are directed through the secretory pathway or targeted to various organelles. Molecules are taken up from the cell's environment through the endocytotic pathway and sorted to different cellular locations. Similarities in sorting have emerged between the secretory and endocytotic pathways. For example, most membrane-associated proteins appear to flow through default pathways unless they are specifically retained or targeted to particular destinations (Hopkins, 1992). Small GTP binding proteins are localized to distinct compartments within both pathways and are involved in regulation of membrane traffic (Novick and Brennwald, 1993). Although more is becoming understood about intracellular trafficking through both the secretory and endocytotic pathways, the mechanisms underlying the sorting decisions are not well characterized.

Internalized ligand/receptor complexes in the endocytotic pathway may be sorted to a variety of destinations. For example, transferrin releases its iron in the acidic environment of the endosome and almost completely recycles back to the cell surface with its receptor (Hopkins and Trowbridge, 1983). In contrast, low density lipoprotein dissociates from its receptor and is degraded in lysosomes while its receptor recycles back to the cell surface (Goldstein et al., 1985). Signaling molecules, such as growth factors, are often degraded along with their receptors down-regulating cellular response to future hormonal stimulation (Carpenter and Cohen, 1976; Stoscheck and Carpenter, 1984).

In the absence of specific targeting, the default pathway for internalized membrane and membrane-associated proteins is recycling back to the cell surface (Hopkins, 1992; Mayor et al., 1993). Fluorescent lipid analogues internalized in fibroblasts were efficiently recycled back to the plasma membrane (Koval and Pagano, 1990). The majority of endocytosed plasma membrane proteins have also been shown to recycle with only a minor fraction being degraded (Burgert and Thilo, 1983; Raub et al., 1986).

Differential endosomal sorting of recycling receptors from dissociated, fluid-phase ligands has been quantitatively studied. Dunn et al.(1989) demonstrated that repeated removal of recycling receptors increases sorting efficiency. The iterative removal of recycling components from the endosomal tubules which possess much higher area-to-volume ratios than the endosomal central vesicle was proposed as a mechanism for the segregation of membrane-associated, recycling components from fluid-phase, lysosomally directed molecules (Dunn et al., 1989; Mayor et al., 1993).

Membrane-associated components are also specifically targeted for degradation, and this is a much less understood phenomenon. Signaling ligand/receptor complexes, such as EGF/receptor complexes, (^1)platelet-derived growth factor/receptor complexes, and colony-stimulating factor/receptor complexes, are often targeted for degradation in lysosomes (Stoscheck and Carpenter, 1984; Guilbert and Stanley, 1986; Sorkin et al., 1991). Other membrane-associated proteins, such as lysosome-associated membrane protein 1, are internalized from the plasma membrane and targeted to lysosomes (Guarnieri et al., 1993). Aggregated receptors (e.g. immune complexes) are also routed to lysosomes (Ukkonen et al., 1986; Mellman and Plutner, 1984).

Linderman and Lauffenburger(1988) proposed that endosomal sorting of membrane-associated receptors is governed by molecular transport out of the central endosomal vesicle into recycling tubules. They postulated that endosomal retention resulting in lysosomal targeting of membrane-associated receptors is modulated by interactions with putative sorting components and that these interactions may be influenced by the receptor's state of occupancy and aggregation.

We have employed EGF and its receptor as a model system with which to investigate the endosomal sorting of membrane-associated receptors. Herbst et al.(1994) demonstrated that the intracellular trafficking of the EGF receptor (EGFR) is regulated by an endosomal apparatus which preferentially recognizes cytoplasmic domain elements of occupied EGFRs compared to unoccupied EGFRs. We have further characterized the endosomal sorting of EGF/receptor complexes using a series of EGFR mutants and demonstrated that lysosomal targeting of occupied EGFRs occurs via endosomal retention that is both specific and saturable (French et al., 1994). These findings are consistent with the theoretical model of endosomal sorting proposed by Linderman and Lauffenburger(1988).

Using members of the EGF family as well as several site-directed recombinant hEGF mutants, we have now investigated how receptor-ligand interactions modulate endosomal sorting. The pH sensitivities of different EGF ligands' binding parameters were measured and correlated with endosomal sorting outcomes. We present evidence that changes in ligand binding at the low endosomal pH levels directly alter the intracellular trafficking of the ligand.


EXPERIMENTAL PROCEDURES

Materials

mEGF, purified from mouse submaxillary glands, was a generous gift from H. S. Wiley (University of Utah). Recombinant hEGF proteins were produced in Escherichia coli and purified with reverse-phase high performance liquid chromatography (Tadaki and Niyogi, 1993). TGFalpha was purchased from Life Technologies, Inc. Ligands were iodinated with I (Amersham) using IODOBEADS following the manufacturer's protocol (Pierce Biochemicals). Free iodine was removed from labeled ligands with an 8-ml column of Sephadex G-15 (Pharmacia). Specific activity of the labeled ligands ranged from 45,000 to 250,000 cpm/ng. B82 mouse fibroblast cells, which lack endogenous EGF receptors, that had been transfected with the wild-type (WT) human EGF receptor were a generous gift of G. N. Gill (University of California at San Diego). Cell surface receptor expression was determined to be 2.4 times 10^5 receptors per cell by Scatchard analysis at 0 °C (Chen et al., 1989). Transfected cells were maintained with 5 µM methotrexate and were grown in Dulbecco's modified Eagle's medium (Flow Laboratories) containing dialyzed 10% calf serum (Sigma). Phenylarsine oxide (PAO) was purchased from Sigma.

Association and Dissociation Rate Constants

B82 cells were grown to confluence in 35-mm tissue culture dishes and were switched to serum-free Dulbecco's modified Eagle's medium with no bicarbonate containing 20 mM HEPES (pH 7.4) and 1 mg/ml bovine serum albumin (D/H/B) 4 to 12 h before experiments. Internalization must be inhibited during association experiments so that the measured association and dissociation with the cell surface are not compromised. Internalization can be inhibited by using temperature or pharmacological manipulations. Experiments performed at 4 °C effectively eliminate internalization (Dunn et al., 1980), but this lower temperature affects the values of association and dissociation rate constants (Lauffenburger and Linderman, 1993). PAO has been shown to be a potent and specific inhibitor of internalization (Low et al., 1981; Gibson et al., 1989). Relative levels of inhibition achieved using low temperature or PAO were investigated in an experiment with three parallel sets of six plates. Two sets experienced no pretreatment while one set was incubated with 0.1 mM PAO at 4 °C for 20 min. All three sets were then incubated with 1.7 nMI-hEGF for 2 h at either 4 °C or 37 °C. Inhibition of internalization was measured by examining the amount of labeled ligand associated with the cell surface compared to the amount internalized. In the absence of any intervention at 37 °C, the ratio of surface-associated to internalized ligand was 0.8 ± 0.3. The plates which were pretreated with 0.1 mM PAO and incubated at 37 °C had a ratio of 15 ± 2, and the plates without pretreatment incubated at 4 °C had a ratio of 44 ± 6. Therefore, PAO pretreatment will significantly inhibit internalization while still allowing measurements of binding parameters to be made at physiologically relevant temperatures. Further controls were performed to determine both the minimal concentration of PAO and incubation time necessary to effectively inhibit internalization in B82 fibroblasts.

Cells used in association experiments were pretreated for 20 min with 0.1 mM PAO. A concentrated 0.1 M PAO stock solution was made in dimethyl sulfoxide and then diluted to the final concentration in WHIPS buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.5 mM MgCl(2), 1 mM CaCl(2), and 1 mg/ml polyvinylpyrrolidone). Following the PAO pretreatment, parallel plates of cells were incubated at 37 °C in 1 ml of 1.7 nM labeled ligand for 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, and 10 min after which they were transferred to 4 °C and rapidly washed five times with 1 ml of ice-cold WHIPS buffer. The cells were washed for 12 min with an acid strip (50 mM glycine-HCl, 100 mM NaCl, 2 mg/ml polyvinylpyrrolidone, pH 3.0) containing 2 M urea to remove surface-bound radioactivity (98% stripping efficiency (Wiley et al., 1991)) and were then solubilized with 2% sodium dodecyl sulfate (SDS) to check that the amount of internalized radioactivity was minimal. A plot of the number of surface-associated ligands per cell versus time was fit with an analytic solution to the following differential equation which describes the interaction between surface receptors and ligands in the absence of internalization and with no ligand depletion (Lauffenburger and Linderman, 1993): dC/dt = k(f)(R - C)L(o) - k(r)C where C, R, L(o), k(f), and k(r) represent the number of surface complexes, the total number of receptors, the ligand concentration, and the association and dissociation rate constants, respectively. To simulate environments at the cell surface or in early endosomes, these experiments were performed either in 20 mM HEPES-buffered Dulbecco's modified Eagle's medium with 1 mg/ml bovine serum albumin at pH 7.4 or in 0.1 M citric acid/phosphate buffer with 1 mg/ml polyvinylpyrrolidone and 135 mM NaCl at pH 6.

Steady-state Sorting Assay

The steady-state endosomal sorting assay described by French et al., 1994 was used to directly monitor endosomal sorting outcomes. Parallel plates of cells were incubated at 37 °C in 2 to 5 mls of D/H/B buffer containing various concentrations (0.008-17 nM) of I-ligand for 2 h to allow the sorting process to reach a steady-state (Wiley et al., 1991, Herbst et al., 1994). After the 2-h incubation, the cells were washed with acid-strip at 4 °C to remove most of the surface-bound ligand (90% stripping efficiency (Wiley et al., 1991)), rinsed twice with 1 ml of phosphate-buffered saline, and returned to 37 °C. The cells were then bathed in 1 ml of D/H/B buffer containing 167 nM unlabeled mEGF to inhibit the rebinding and reinternalization of recycled labeled ligand. The medium was collected from parallel plates at 0, 0, 5, 5, 10, and 15 min. Cells were rinsed five times with 1 ml of ice-cold WHIPS buffer, washed with acid-strip containing 2 M urea to remove the surface-bound radioactivity, and then solubilized with 2% SDS to collect the internalized radioactivity. A mass balance for each time point was calculated by adding the amount of radioactivity internalized, released into the medium, and associated with the surface. Cell number per plate was determined with parallel plates using a hemocytometer. Nonspecific binding was determined to be less than 2% of total binding in B82 cells (Wiley et al., 1991). Nonspecific internalization of fluid-phase ligand was investigated by comparing the amounts of labeled ligand which nontransfected B82 cells and B82 cells transfected with the EGFRs internalized after 2 h and was generally found to be negligible (less than 2%) except at high concentrations (e.g. for mEGF at 17 nM, nonspecific internalization accounted for 8% of the internalized ligand) and for the low affinity ligands (e.g. for our lowest affinity ligand, Y13G, nonspecific internalization accounted for approximately 20% of the internalized ligand at all concentrations).

Degradation and Recycling

Degraded and intact (recycled) radiolabeled ligands in the medium were separated using an 8-ml column of Sephadex G-15 and quantified with a Packard 5000 series -counter. The first elution peak represented undegraded EGF, while the second peak was degraded EGF composed mainly of monoiodotyrosine (Carpenter and Cohen, 1976; Wiley and Cunningham, 1982). The sorting fraction or percent recycled was defined as the radioactivity in the first peak divided by the total radioactivity collected from the column. Sorting fractions at each of the three time points (5, 10, and 15 min) were averaged to calculate the overall sorting fraction for each experiment. The observed degradation rate was calculated by quantifying the radioactivity in the second peak, dividing by the chase time to get the number of molecules degraded per min per cell, and averaging over each of the time points. Values of the observed degradation rate constants were calculated by dividing the observed degradation rate by the number of intracellular ligand molecules per cell.


RESULTS

Association Experiments

TGFalpha, mEGF, and hEGF are all members of the EGF family and bind to the EGFR. Association experiments were performed with these ligands and several site-directed recombinant hEGF mutants to determine how their association and dissociation rate constants were affected by changes in pH. Following pretreatment with PAO, cells were incubated at 37 °C with 1.7 nMI-ligand in buffer either at pH 7.4 or pH 6 mimicking the different pH environments on the cell surface and in early endosomes (Mellman et al., 1986; Sorkin et al., 1988). The amount of surface-bound ligand was monitored as a function of time. A plot of the number of surface-associated ligands per cell versus time was fit to determine the association and dissociation rate constants (Lauffenburger and Linderman, 1993). Fig. 1presents composite association curves for three experiments with hEGF, mEGF, and TGFalpha at pH 7.4 and pH 6. The association rate constant dominated the initial slope of these curves while the ratio of the dissociation to association rate constants controlled the shape of the curve after several minutes.


Figure 1: Composite curves of association experiments with mEGF, hEGF, and TGFalpha. After a 20-min pretreatment with 0.1 mM phenylarsine oxide at 4 °C to inhibit internalization, parallel plates of B82 fibroblasts were incubated at 37 °C with 1.7 nMI-ligand either in D/H/B buffer (pH 7.4) or citric acid/phosphate buffer (pH 6.0). At time intervals up to 10 min, plates of cells were washed at 4 °C with an acid-strip (pH 3.0) containing 2 M urea to collect the surface-associated ligands. Composite plots of the surface-associated ligands per cell versus time from three experiments are shown for mEGF (), hEGF (circle), and TGFalpha (box). (Filled and open symbols represent measurements at pH 7.4 and pH 6, respectively.)



Average individual rate constants at pH 7.4 and 6 were determined from a minimum of three separate experiments for each of the different ligands (Table 1). Individual association and dissociation rate constants were used to calculate equilibrium dissociation constants (K(d)) for the different ligands at both pH 7.4 and pH 6 (Fig. 2A). The ratio of K(d) values in pH 6 buffer to K(d) values in pH 7.4 buffer provided a measurement of the pH sensitivity of each ligand's binding constants. Although mEGF, hEGF, and TGFalpha had roughly equivalent K(d) values at pH 7.4, the ratio of hEGF K(d) values in pH 6 and pH 7.4 environments was 4.5 times greater than that of mEGF, while the ratio for TGFalpha was 9 times greater than that of mEGF (Fig. 2B).




Figure 2: Equilibrium dissociation constant values at pH 7.4 and pH 6. A, individual association and dissociation rate constants tabulated in Table 1were used to calculate equilibrium dissociation constants (K) at pH 7.4 (box) and pH 6 (bullet). B, pH sensitivity of the equilibrium dissociation constants for the different ligands is represented by their ratio of K values in pH 6 buffer to Kvalues in pH 7.4 buffer.



Steady-state Sorting of mEGF, hEGF, and TGFalpha

Steady-state sorting experiments were performed with mEGF, hEGF, and TGFalpha. Sorting events were isolated from surface binding effects by plotting the sorting fractions versus the number of intracellular ligands per cell rather than the incubation concentration. mEGF, hEGF, and TGFalpha exhibited different sorting patterns as the number of intracellular ligands per cell was varied over 3 orders of magnitude (Fig. 3). The fraction of mEGF which was recycled increased from approximately 45% to 80% as the number of intracellular ligands per cell rose from about 400 to 100,000. In striking contrast, the fraction of TGFalpha which was recycled decreased slightly from 55% to 48% over the same range of intracellular ligand levels. The sorting outcome for hEGF appeared fairly insensitive to changes in intracellular ligand levels, and a relatively constant fraction (56-59%) of hEGF was recycled over the same range of intracellular ligand levels.


Figure 3: Different steady-state sorting outcomes of several members of EGF family. Plates of confluent B82 cells were incubated for 2 h at 37 °C in various concentrations (0.008-17 nM) of I-mEGF (), I-hEGF (bullet), or I-TGFalpha (box). The cells were then washed with acid-strip (pH 3) at 4 °C to remove most of the surface-bound ligand, returned to 37 °C, and chased with 167 nM unlabeled mEGF. The medium was collected from parallel plates at 0, 5, 10, and 15 min. Cells were washed with acid-strip containing 2 M urea to remove the surface-bound radioactivity and were solubilized with 2% SDS to collect the internalized radioactivity. Degraded and intact (recycled) radiolabeled ligands in the medium were separated using gel filtration. Sorting fraction represents the fraction of the internalized ligand which is recycled and is defined as the radioactivity in the first elution peak divided by the total radioactivity collected from the gel filtration column. The sorting fractions at each of the three time points (5, 10, and 15 min) were averaged to calculate the overall sorting fraction for each experiment.



Binding affinities and sorting fractions can be compared to receptor down-regulation levels to help provide some basic insights into trafficking dynamics. We found that 27%, 34%, and 65% of the original numbers of surface receptors remain following a 2-h incubation in 100 ng/ml mEGF, hEGF, and TGFalpha, respectively. These measurements correspond well with down-regulation results in B82 cells reported by Chen et al.(1989) for mEGF and by Ebner and Derynck(1991) for hEGF and TGFalpha. Since EGFRs internalize at similar rates when bound by mEGF, hEGF, or TGFalpha, the observed differences in down-regulation imply that slightly more receptors recycle when internalized with hEGF than with mEGF, and significantly more receptors recycle when internalized with TGFalpha than with either mEGF or hEGF. These findings also correlate well with the extent of endosomal receptor/ligand complex dissociation measured for the various ligands since unoccupied EGFRs predominantly recycle (Herbst et al., 1994).

Steady-state Sorting of hEGF and hEGF Mutants

The intracellular trafficking of several site-directed recombinant hEGF mutants was also investigated. Tyrosine 13 is a highly conserved amino acid in EGF-like proteins and appears to play an important role in contributing to the hydrophobic binding site environment of EGF and its receptor (Tadaki and Niyogi, 1993). Glutamate 40 is involved in binding by virtue of its proximity to the crucial arginine 41 residue (Campion et al., 1992). The binding parameters of several mutants generated at these residues were evaluated. E40A, Y13H, and Y13G retained approximately 5%, 10%, and 2% of the affinity of hEGF at pH 7.4, respectively (Table 1). These mutants had larger equilibrium dissociation constants than hEGF in pH 6 environments (Fig. 2).

Steady-state sorting experiments were performed with these hEGF mutants. Differences in surface binding (pH 7.4) were normalized by plotting the sorting fractions versus the number of intracellular ligands per cell rather than the incubation concentration. This approach isolated intracellular sorting behavior from surface binding and internalization events and allowed the sorting behavior of different ligands to be compared in cells with similar amounts of intracellular ligands. Sorting patterns of the mutants were different from the sorting pattern of hEGF (Fig. 4) and showed a slightly negative relationship to increasing intracellular ligand levels per cell analogous to TGFalpha's sorting behavior, although their actual sorting fractions were higher than the sorting fractions of TGFalpha.


Figure 4: Steady-state sorting patterns of hEGF and several hEGF mutants. Plates of confluent B82 cells were incubated for 2 h at 37 °C in various concentrations (0.008-17 nM) of I-Y13G (), I-Y13H (), or I-E40A (up triangle) followed by a chase with excess unlabeled ligand (167 nM). Degraded and intact (recycled) ligands in the medium were separated with gel filtration, and sorting fractions were calculated as described in the legend to Fig. 3. The sorting pattern of hEGF (bullet) is shown for comparison.



Observed Degradation Rate Constants

The observed degradation rate constant (k(h)) is a lumped kinetic parameter describing the proteolytic and transport processes ligands undergo as they move through the degradation pathway. Values of k(h) were measured for mEGF, hEGF, and TGFalpha (Fig. 5). Parallel plates of fibroblasts were first incubated for 2 h in labeled ligand at 37 °C and then chased with an excess of unlabeled ligand. The accumulation of degradation products in the medium was monitored at 5-, 10-, and 15-min intervals during the chase, and the numbers of ligand molecules degraded per min per cell for each of the time points were averaged. Values of observed degradation rate constants were calculated by dividing the number of ligand molecules degraded per min per cell by the number of intracellular ligand molecules per cell. Values of k(h) for both mEGF and hEGF decreased as functions of increasing intracellular ligand levels. In contrast, the values of k(h) for TGFalpha remained relatively constant.


Figure 5: Observed degradation rate constants of mEGF, hEGF, and TGFalpha as functions of the number of intracellular ligands per cell. Observed degradation rate constants of mEGF (), hEGF (bullet), and TGFalpha (box) were determined for the steady-state experiments shown in Fig. 3. The observed degradation rate was determined by quantifying the radioactivity in the second gel filtration peak, dividing by the chase time to compute the number of molecules degraded per min per cell, and averaging over each of the time points. Values of the observed degradation rate constants were calculated by dividing the observed degradation rate by the number of intracellular ligand molecules per cell.



Values of k(h) were also measured for the hEGF mutants and compared to the values of k(h) for hEGF (Fig. 6). Values of k(h) for the hEGF mutants were insensitive to increasing numbers of intracellular ligand molecules and were lower than hEGF k(h) values. Although the values of k(h) for hEGF mutants and TGFalpha were all fairly insensitive to changes in intracellular ligand levels, the magnitudes of the k(h) values of the hEGF mutants were significantly lower than those for TGFalpha.


Figure 6: Observed degradation rate constants of hEGF and several hEGF mutants. Observed degradation rate constants of Y13G (), Y13H (), and E40A () were determined for the steady-state experiments shown in Fig. 4. Calculation of observed degradation rate constants was performed as described in the legend to Fig. 5. Observed degradation rate constants of hEGF (bullet) are shown for comparison.




DISCUSSION

Several examples exist of receptor mutations which alter receptor/ligand interactions and consequently change the intracellular trafficking of receptor/ligand complexes. A deletion mutant of the LDL receptor was constructed which did not release its ligand in the low pH environment of the endosome and consequently was rapidly degraded rather than recycled (Davis et al., 1987). A receptor mutant with similar abnormal ligand binding and trafficking properties was later found in a patient with familial hypercholesterolemia (Miyake et al., 1989). Another example is a mutant insulin receptor isolated in a patient with severe insulin-resistant diabetes (Kadowaki et al., 1988). This receptor was characterized and found not to release insulin efficiently in the acidic environment of the endosome. This failure to release its ligand resulted in the receptor being degraded rather than recycled (Kadowaki et al., 1990).

We have investigated how different ligand properties influence the endosomal sorting of internalized EGF/receptor complexes. Several members of the EGF family are sorted quite differently in the endosome even though they are all internalized with the EGF receptor. The association and dissociation rate constants of these ligands measured at pH 6 are useful in interpreting the different endosomal sorting outcomes. Coupling association and steady-state sorting experiments yields increased understanding of how ligand properties directly affect the intracellular trafficking of the ligand and its receptor.

mEGF, hEGF, and TGFalpha are members of the EGF family which bind to EGFRs with similar affinity at pH 7.4, but their binding is differentially affected by the low pH endosomal environment (Fig. 2). For example, the dissociation rate constant of TGFalpha was 9 times larger at pH 6 than at pH 7.4 while the dissociation rate constants of mEGF and hEGF were only 3 to 4 times larger. pH sensitivity of the binding parameters was assessed using a ratio of equilibrium dissociation constants in pH 6 buffer to the equilibrium dissociation constants in pH 7.4 buffer. The ratio of hEGF K(d) values in pH 6 and pH 7.4 buffers was 4.5 times greater than that of mEGF, while the ratio for TGFalpha was 9 times greater than that of mEGF. The large difference in the pH sensitivity of binding between mEGF and TGFalpha correlated well with experimental data reported by other investigators. Using a permeabilization technique, Sorkin et al.(1988) found that the majority of internalized mEGF molecules remained complexed with receptors. In contrast, Korc and Finman (1989) and Ebner and Derynck(1991) observed that TGFalpha dissociated within the endosome to a much greater extent than EGF.

These three ligands exhibited quite different sorting patterns as the numbers of intracellular ligands per cell were varied over 3 orders of magnitude (Fig. 3). The affinity of mEGF for the EGFR is fairly insensitive to endosomal pH, and the majority of mEGF remains bound to its receptor. Correspondingly, the fraction of internalized mEGF which was recycled rose from 45% to 80% as the increasing number of intracellular complexes saturated the endosomes' ability to target complexes to the lysosomes for degradation (French et al., 1994). In contrast, the affinity of TGFalpha for the EGFR is very sensitive to the lower pH environment in the endosome, and TGFalpha is largely dissociated from its receptor. Its sorting pattern is distinctly different from the saturating behavior observed with mEGF and may simply reflect the partitioning of fluid-phase ligands. The sorting behavior of hEGF is intermediate between the two extremes of ligands almost completely complexed with receptors (mEGF) and ligands almost completely dissociated from receptors (TGFalpha). This finding correlates well with hEGF's intermediate sensitivity to lower pH levels and suggests that the sorting outcomes of hEGF may represent the integration of many short-lived interactions between the ligand and its receptor. These three EGF ligands demonstrate that at high intracellular ligand concentrations a 5- to 10-fold increase in the binding affinity's pH sensitivity can decrease the percentage of the intracellular ligand that is recycled from 80% down to 50%.

Observed degradation rate constants measured for mEGF, hEGF, and TGFalpha also illustrate that the intracellular trafficking of EGF ligands may be directly influenced by the differing pH sensitivities of their binding properties. The observed degradation rate constant represents a lumped kinetic parameter describing the rate at which labeled ligand moves from early endosomes to lysosomes, is degraded, and is transported out of the cell. Values of k(h) for mEGF decreased as a function of increasing intracellular ligand levels. This trend corresponds well with the mEGF sorting fractions and supports the hypothesis that endosomal targeting of mEGF complexes to degradation is saturated at high intracellular ligand levels. In contrast, values of k(h) for TGFalpha remained relatively constant suggesting that the degradation of TGFalpha is the result of a nonspecific process and appears to be simply proportional to the number of internalized TGFalpha molecules. The values of k(h) for hEGF fell between the values for mEGF and TGFalpha indicating that some specific endosomal targeting of hEGF was being saturated but not to the same extent as for mEGF. Both the steady-state endosomal sorting patterns and observed degradation rate constants of these EGF ligands illustrate how different receptor-ligand interactions in the low pH endosomal environment can change the intracellular trafficking of the ligands.

The differences observed in trafficking between hEGF and mEGF may be related to sequence differences between these ligands. hEGF, unlike mEGF, shares several histidine residues (10 and 16) with TGFalpha (Carpenter and Wahl, 1990) conferring a greater sensitivity to changes in pH. Binding differences between mEGF and hEGF at the low pH levels found in endosomes result in altered intracellular trafficking as evidenced by different steady-state sorting patterns and observed degradation rate constants.

Single amino acid substitutions in hEGF result in altered intracellular trafficking which correlates with changes in the binding properties of the mutants at pH 6. The hEGF mutants have dissociation rate constants at pH 6 that are roughly 3 times larger than that of hEGF. When the number of intracellular ligands per cell is low, 10-15% more of the internalized mutant hEGF is recycled than hEGF. Degradation rate constants for the mutants were about half the magnitude of hEGF values at low intracellular ligand levels and were much less sensitive to changes in intracellular ligand levels. These differences between the intracellular trafficking of hEGF mutants and hEGF, which were most distinct at low intracellular ligand levels, illustrate the direct relationship between the altered binding of these mutants at endosomal pH levels and sorting outcomes. Although the mutants' binding properties at pH 6 were similar to those of TGFalpha, larger percentages of the hEGF mutants were recycled, and their k(h) values were significantly lower at all intracellular ligand levels than TGFalpha.

Interpretation of the differences observed in steady-state sorting measurements between the low affinity hEGF mutants and hEGF may be complicated by several factors. The sorting fraction reflects not only accumulation of degraded ligand and dissociated recycled ligand in the medium but also dissociation of residual, noninternalized ligand from the cell surface. This potential distortion was minimized by using an acid-strip during the experiment to remove most of the noninternalized, surface-associated ligand before the measurements were made, and we have previously established that the experimental sorting fraction does accurately represent the true percentage of recycled mEGF (French et al., 1994). The low affinity mutants dissociate from the surface faster than mEGF (k(r) = 0.4 to 1.2 min for hEGF mutants, k(r) = 0.3 min for mEGF). This may skew measurements of the low affinity mutants' sorting fractions artificially higher as any residual, noninternalized ligand will dissociate more rapidly. Another factor which may distort the sorting fractions of the mutants is that a larger fraction of the endosomal ligand is nonspecifically internalized. This effectively raises the endosomal concentration of ligand relative to the number of intracellular receptors and may alter the dynamics of the interactions within endosomes skewing the sorting fraction lower. However, observed degradation rate constants should not be influenced by increased dissociation from the surface during the measurements, and the impact of increased nonspecific internalization should be to increase degradation rate constants. Measured degradation rate constants of the low affinity mutants were significantly lower than hEGF values demonstrating that the intracellular trafficking of the hEGF mutants is indeed altered compared to hEGF.

These experiments show that changes in ligand binding properties can quantitatively modify endosomal sorting outcomes. Ligands of the EGF family, which are all internalized bound to the EGFR, undergo different intracellular processing that may account for differences that have been observed in cellular response (Schreiber et al., 1986; Korc et al., 1987; Brenner et al., 1989). Single amino acid substitutions can modulate endosomal sorting outcomes by changing the growth factor's binding properties at low pH levels. This suggests that ligands may be genetically engineered to achieve desired changes in intracellular trafficking and thereby potentially modify cellular response. A more rigorous understanding of how ligand properties govern trafficking may facilitate improved therapeutic interventions in areas such as wound healing, cancer treatment, and tissue regeneration (Lauffenburger, 1994).


FOOTNOTES

*
This work was funded by the National Science Foundation Biotechnology Program and the Johnson & Johnson Focused Giving Program (to D. A. L.) and a National Science Foundation Subcontract DE-AC05-84OR21400 to the United States Department of Energy with Martin Marietta Energy Systems (to S. K. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; m, mouse; h, human; TGF, transforming growth factor; PAO, phenylarsine oxide.


ACKNOWLEDGEMENTS

We gratefully acknowledge very helpful discussions with H. Steve Wiley (University of Utah).


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