(Received for publication, September 13, 1994; and in revised form, December 1, 1994)
From the
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
(TGF
) 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 TGF
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.
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, ()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.
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,
1 mM CaCl
, 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
(R - C)L
- k
C where C, R, L
, k
, and k
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.
Figure 1:
Composite curves
of association experiments with mEGF, hEGF, and TGF. 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 nM
I-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 (
), and TGF
(
). (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) for the different ligands at both pH 7.4 and
pH 6 (Fig. 2A). The ratio of K
values in pH 6 buffer to K
values in pH 7.4
buffer provided a measurement of the pH sensitivity of each
ligand's binding constants. Although mEGF, hEGF, and TGF
had
roughly equivalent K
values at pH 7.4, the ratio
of hEGF K
values in pH 6 and pH 7.4 environments
was 4.5 times greater than that of mEGF, while the ratio for TGF
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 (
) and pH 6 (
). 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 K
values in pH 7.4
buffer.
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 (
), or
I-TGF
(
).
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 TGF,
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 TGF
. Since EGFRs
internalize at similar rates when bound by mEGF, hEGF, or TGF
, 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 TGF
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 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 TGF's sorting behavior,
although their actual sorting fractions were higher than the sorting
fractions of TGF
.
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 (
) 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 (
) is shown for
comparison.
Figure 5:
Observed degradation rate constants of
mEGF, hEGF, and TGF as functions of the number of intracellular
ligands per cell. Observed degradation rate constants of mEGF (
),
hEGF (
), and TGF
(
) 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 were also
measured for the hEGF mutants and compared to the values of k
for hEGF (Fig. 6). Values of k
for the hEGF mutants were insensitive to
increasing numbers of intracellular ligand molecules and were lower
than hEGF k
values. Although the values of k
for hEGF mutants and TGF
were all fairly
insensitive to changes in intracellular ligand levels, the magnitudes
of the k
values of the hEGF mutants were
significantly lower than those for TGF
.
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
(
) are shown for comparison.
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 TGF 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 TGF
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
values in pH 6
and pH 7.4 buffers was 4.5 times greater than that of mEGF, while the
ratio for TGF
was 9 times greater than that of mEGF. The large
difference in the pH sensitivity of binding between mEGF and TGF
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 TGF
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 TGF for the EGFR is very sensitive to
the lower pH environment in the endosome, and TGF
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 (TGF
). 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
TGF 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
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
for TGF
remained relatively constant suggesting that the degradation of
TGF
is the result of a nonspecific process and appears to be
simply proportional to the number of internalized TGF
molecules.
The values of k
for hEGF fell between the values
for mEGF and TGF
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 TGF
(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 TGF, larger percentages of the hEGF mutants were
recycled, and their k
values were significantly
lower at all intracellular ligand levels than TGF
.
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 = 0.4 to 1.2
min
for hEGF mutants, k
= 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).