From the Division of Hematology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
¶ Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
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ABSTRACT |
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The cation-independent mannose
6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II
receptor) undergoes constitutive endocytosis, mediating the
internalization of two unrelated classes of ligands, mannose
6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like
growth factor II (IGF-II). To determine the role of ligand valency in
M6P/IGF-II receptor-mediated endocytosis, we measured the
internalization rates of two ligands, The mannose 6-phosphate/insulin-like growth factor II receptor
(M6P/IGF-II receptor)1 is a
type I transmembrane glycoprotein that cycles through the Golgi,
endosomes, and the plasma membrane to carry out its role in the
biogenesis of lysosomes and in the clearance of the polypeptide insulin-like growth factor II (IGF-II) (1, 2). In the Golgi, the
receptor binds newly synthesized acid hydrolases modified with mannose
6-phosphate (Man-6-P) residues on their asparagine-linked oligosaccharides and transports them to endosomes via clathrin-coated vesicles (3-5). The acid hydrolases are released in the acidified endosome and then packaged into lysosomes while the receptor either returns to the Golgi to bind another ligand or moves to the plasma membrane (6, 7). At the plasma membrane, the M6P/IGF-II receptor
mediates internalization of Man-6-P-containing ligands and IGF-II (3,
5, 8).
The interactions of IGF-II and Man-6-P-containing ligands with the
M6P/IGF-II receptor have been characterized in several studies (8-12).
The extracellular portion of the M6P/IGF-II receptor contains 15 homologous repeating domains of ~147 amino acids each (13). Domains 3 and 9 (numbering from the amino terminus) each bind 1 mol of Man-6-P,
and the single IGF-II-binding site has been mapped to domain 11 in the
extracellular region (14-16). Man-6-P residues do not inhibit binding
of IGF-II to the receptor, verifying that the two ligand-binding sites
are distinct. However, proteins containing Man-6-P residues do compete
with IGF-II for receptor binding, and IGF-II can inhibit binding of
lysosomal enzymes to the receptor (8-10, 17). In neither case is the
competition complete, and the most plausible explanation is that the
inhibition is due to steric hindrance.
Although the M6P/IGF-II receptor has been shown to be constitutively
internalized from the cell surface, it is not clear whether ligand
binding influences the trafficking of the receptor. It has been
reported that in the absence of ligand, the M6P/IGF-II receptor
accumulates in the Golgi, whereas the addition of lysosomotropic agents
that prevent the release of ligand from the receptor in endosomes
results in an accumulation of the receptor in these organelles
(18-20). Other investigators have found that constitutive trafficking
of the M6P/IGF-II receptor continues under these conditions (21, 22).
Together, these data are consistent with the concept that ligand
binding modulates the rate of receptor trafficking. Thus, the absence
or presence of bound ligand may regulate the trafficking from specific
compartments, resulting in a shift in the steady-state distribution of
the receptor. However, none of these studies have actually determined
the kinetics of receptor trafficking.
In this study, we have compared the internalization of
Materials--
Recombinant human IGF-II was purchased from
Bachem California; IGF-II-(del 1-6) from Upstate Biotechnology, Inc.,
Na125I from Amersham Pharmacia Biotech; lactoperoxidase
from Calbiochem; Man-6-P from Sigma; and Lipofectin and G418 from Life
Technologies, Inc. Other reagent-grade chemicals were from standard
suppliers. The bivalent ligand
(Ac-Thr-[ Transfection of Receptor-deficient Cells--
The transfection
of the M6P/IGF-II receptor-negative mouse L cell line
(L(Rec Purification and Iodination of Iodination of IGF-II--
10 µg of IGF-II was iodinated using
the lactoperoxidase method described for Iodination of IGF-II-(del 1-6)--
IGF-II-(del 1-6) was
iodinated by coating an Eppendorf tube with 50 µg of IODO-GEN
(Pierce) and adding 5 µg of IGF-II-(del 1-6) and 1 mCi of
Na125I. The mixture was incubated at room temperature for 3 min and then loaded onto a PD10 column (Amersham Pharmacia Biotech)
pre-equilibrated in PBS plus 1% BSA. Fractions containing the first
peak were pooled and stored at 4 °C. The typical activity assuming
complete protein recovery was 108 cpm/µg of protein.
Rapid Endocytosis Assays--
Cells were grown to confluence in
12-well plates. The cells were rinsed twice in ice-cold PBS and 1%
BSA, and ligand was added in cold Preparation of the M6P/IGF-II Receptor--
110 g of fresh
bovine liver was minced and blended in a Waring blender in 200 ml of
ice-cold extraction buffer (50 mM imidazole (pH 7), 150 mM NaCl, 5 mM sodium Determination of the Stokes Radius--
A Superose 6 FPLC column
was equilibrated in filtered and degassed wash buffer. Protein
standards were run and detected by absorbance at 280 nm. The
Kd, defined as (Ve Determination of Sedimentation Coefficients--
Continuous
6-21% sucrose gradients (4.8 ml) were prepared in wash buffer and
allowed to equilibrate at 4 °C for 1 h. Samples were loaded
onto the gradients, and the gradients were centrifuged for 4 h at
237,000 × gav in an SW 55Ti rotor.
Fractions (240 or 120 µl) were collected from the top of the
gradient. Refractive indices of each fraction were measured to
determine linearity of the gradients. Fractions were analyzed by
SDS-polyacrylamide gel electrophoresis following chloroform/methanol
precipitation or by IGF-II and
By contrast, Intermolecular Cross-linking of M6P/IGF-II Receptors Is Responsible
for the Ligand-induced Increase in the Internalization
Rate--
The second approach to distinguish intra- from intermolecular
cross-linking utilized cells expressing a mutant receptor that has only
a single functional Man-6-P-binding site. The Man-6-P binding of domain
3 was abolished by substituting an alanine for Arg-435 (14, 25). The
full-length receptor containing this mutation was transfected into
L(Rec
The ability of Dom3ala cells to internalize
Characterization of the Oligomeric State of the Receptor--
We
next determined the state of oligomerization of purified M6P/IGF-II
receptor in both the presence and absence of
To determine the effect of
Using these values for the Stokes radius and sedimentation coefficient,
along with a calculated partial specific volume of 0.73, the molecular
weight for the complex was determined to be 912,000. This value is
within 2% of the predicted value of 928,000 for a complex composed of
two M6P/IGF-II receptors and one
In other experiments, the receptor was incubated with saturating
concentrations of either IGF-II or the Man-6-P-containing peptide and
then subjected to FPLC gel filtration. In both instances, the elution
positions of the receptor were identical to that of the receptor alone,
indicating that neither compound induced dimerization of the receptor
(data not shown).
The data presented in this study show that the M6P/IGF-II
receptor internalizes How, then, does cross-linking of the M6P/IGF-II receptor by
Dimerization of receptor tails, in a simplistic sense, would lead to a
doubling of signals, which presumably would interact more effectively
with the adaptor proteins localized in coated pits. Although the
analysis of the solubilized receptor indicates that it is a monomer, it
is not possible to directly extrapolate this finding to the
membrane-bound form of the receptor since a loosely associated dimer in
the membrane might fall apart upon solubilization. This is relevant to
the rapid internalization assays. In these experiments,
Recently, the three-dimensional structure of the extracellular domain
of the cation-dependent Man-6-P receptor has been solved by
Roberts et al. (39). The domain crystallized as a dimer, with the single Man-6-P-binding site of each monomer being oriented in
the same direction. The authors modeled the interaction of the
cation-dependent Man-6-P receptor dimer with
Ligand-induced internalization of plasma membrane signaling receptors,
such as the epidermal growth factor receptor, provides a mechanism for
the down-regulation of these receptors and the termination of the
signaling. However, it seems unlikely that this would be the
physiologic function of this process in the case of the M6P/IGF-II
receptor, which cycles constitutively between the plasma membrane,
endosomes, and the Golgi. The major role of the receptor at the cell
surface is to bind and internalize IGF-II, and the receptor probably
does not encounter significant amounts of acid hydrolases at this
location. On the other hand, this mechanism could impact on the
kinetics of sorting of the M6P/IGF-II receptor in the
trans-Golgi network. If the AP-1 adaptor complex of the
Golgi clathrin-coated pits interacts preferentially with receptor
associated with an acid hydrolase ligand, then free receptor would be
present in the trans-Golgi network for a somewhat longer
period of time and have more opportunity to bind ligand. As a
consequence, sorting efficiency would be enhanced, particularly if the
amount of the M6P/IGF-II receptor in the trans-Golgi network is limiting. Conversely, sorting at the endosome would be most efficient if the receptor exited this compartment faster after ligand
release. In this case, the sorting signal for targeting from the
endosome to the trans-Golgi network would be exposed optimally upon release of ligand. It has been reported that cells devoid of acid hydrolase ligands exhibit an accumulation of M6P/IGF-II receptors in the Golgi, whereas cells in which ligand dissociation is
blocked accumulate M6P/IGF-II receptors in endosome-like structures (18-20). Although several studies have shown that constitutive trafficking of the receptor continues under these circumstances (21,
22), these experiments did not exclude the possibility that ligand
occupancy modulates the rate of receptor movement between compartments
and therefore the steady-state level at each station. Our results show
that receptor dimerization induced by multivalent ligands does alter
the kinetics of internalization of the M6P/IGF-II receptor at the
plasma membrane, and this process could potentially influence receptor
movement at other sites as well.
-glucuronidase (a homotetramer
bearing multiple Man-6-P moieties) and IGF-II. We found that
-glucuronidase entered the cell ~3-4-fold faster than IGF-II.
Unlabeled
-glucuronidase stimulated the rate of internalization of
125I-IGF-II to equal that of
125I-
-glucuronidase, but a bivalent synthetic tripeptide
capable of occupying both Man-6-P-binding sites on the M6P/IGF-II
receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize
-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single
Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized
and purified in Triton X-100 was present as a monomer, but association
with
-glucuronidase generated a complex composed of two receptors and one
-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that
intermolecular cross-linking of the M6P/IGF-II receptor occurs upon
binding of a multivalent ligand, resulting in an increased rate of internalization.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucuronidase, a homotetramer with multiple phosphorylated
oligosaccharides, with that of IGF-II. We found that the initial rate
of internalization of
-glucuronidase is much more rapid than that of
IGF-II, providing direct evidence that a multivalent ligand enhances
the rate of movement of the receptor. Furthermore, we present data that
the mechanism of this effect is due to dimerization of the receptor.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-D-Man-6-P(
1-2)
-D-Man]-Lys-(ABz)-Thr-[
-D-Man-6-P(
1-2)
-D-Man]-NH2) was kindly provided by Dr. K. Bock (Carlsberg Laboratory, Copenhagen, Denmark) (23).
)) with constructs encoding the wild-type receptor
and a mutant receptor with a 29-amino acid cytoplasmic tail to give the
Cc2 and 344 cell lines, respectively, has been previously described (5,
24). The Dom3ala cell line expressing a receptor with
an R435A mutation was generated as described (25).
-Glucuronidase--
Human
-glucuronidase was purified from the secretions of 13.2.1 mouse L
cells as described previously (24). This cell line, which has been
engineered to secrete large amounts of
-glucuronidase, was kindly
provided by Dr. William Sly (St. Louis University). For iodination, 30 µg of human
-glucuronidase was brought to 500 µl in PBS (pH
7.4); 2.5 µl of 1.86 µM lactoperoxidase, 1 mCi of
Na125I, and 2 µl of 0.5 nM hydrogen peroxide
were added to start the reaction. After 3 min at room temperature,
another 2 µl of 0.5 nM hydrogen peroxide was added, and
after an additional 3 min, the reaction was stopped by adding 200 µl
of quench solution (1 M NaCl, 100 mM NaI, 50 mM NaPO4 (pH 7.5), 1 mM
NaN3, 2 mg/ml BSA, and 1 mg/ml protamine sulfate). The
quenched reaction mixture was loaded onto a 1-ml M6P/IGF-II receptor
affinity column (26) equilibrated in PBS and 0.1% BSA. After extensive
washing, the
-glucuronidase was eluted with PBS and 0.1% BSA
containing 10 mM Man-6-P. The peak fractions were pooled
and dialyzed against PBS to remove the Man-6-P, and the
125I-
-glucuronidase was stored at 4 °C. The typical
specific activity assuming complete protein recovery was
106 to 107 cpm/µg of protein.
-glucuronidase. After
quenching, the reaction mixture was loaded onto a Sephadex G-25 column
equilibrated in PBS and 0.1% BSA. Three peaks of radioactivity were
observed and subjected to trichloroacetic acid precipitation. The
second peak was found to be 98% trichloroacetic acid-precipitable and
contained monomeric IGF-II. The iodinated ligand was stored at 4 °C.
The typical specific activity assuming complete protein recovery was 107 cpm/µg of protein.
-minimal Eagle's medium and 2%
BSA (0.5 ml/well). 125I-IGF-II was added to a 2 nM final concentration, and
125I-
-glucuronidase was typically added to a final
concentration of 0.12 nM. The plates were then floated on
an ice water bath for 30 min. Unbound ligand was removed, and the wells
were rapidly washed five times with 1 ml of ice-cold PBS and 1% BSA.
To the three wells used for the 0 time point was added 1 ml of ice-cold stop/strip solution (SSS; 0.2 M acetic acid (pH 3.5) and
0.5 M NaCl). The plate was then floated in a 37 °C water
bath, and 0.5 ml of
-minimal Eagle's medium prewarmed to 37 °C
was quickly added to the remaining wells to initiate internalization.
At each stopping point, the
-minimal Eagle's medium in the well was
removed to a tube for counting, and 1 ml of cold SSS was added. At the end of the experiment (usually 3 min), the plate was removed from the
water bath, and the surface-bound ligand was stripped from each well by
incubation for a total of 10 min in ice-cold SSS (1 ml for 5 min,
twice) and counted. The cells were then solubilized in 0.1 N NaOH (1 ml, twice) and counted. The M6P/IGF-II
receptor-negative cell line was used as a control for non-receptor
binding of 125I-IGF-II and
125I-
-glucuronidase. The sum of the labeled ligand
remaining on the cell surface at the end of the internalization
experiment (receptor-bound ligand) and the internalized ligand was used
as a measure of the maximum potential internalization. The fraction of
this value internalized at each time point was calculated and plotted.
This calculation method was used to exclude the contributions of a
specific, non-M6P/IGF-II receptor binding site for IGF-II observed in
all the cell lines, including the receptor-negative cells. This site
has a lower affinity for IGF-II than the M6P/IGF-II receptor, and with
incubation at 37 °C, the ligand is released into the medium rather
than internalized. This calculation method was used for both IGF-II and
-glucuronidase binding studies.
-glycerophosphate, 2%
Triton X-100, 0.25% deoxycholate, 10 mM EDTA, 50 µg/ml
leupeptin, 50 µg/ml aprotinin, 50 µg/ml trypsin inhibitor, and 50 µg/ml phenylmethylsulfonyl fluoride) for ~10 s, four times. The
homogenate was centrifuged at 30,000 × g for 30 min,
and the supernatant was poured through cheesecloth. A ~15-ml packed
volume of phosphopentamannose-agarose beads (27) was washed with
extraction buffer without protease inhibitors and added to the
supernatant. Receptor binding was allowed to occur for 30 min at
4 °C while rocking. The beads were collected by filtering over a
coarse Buchner funnel, and the agarose beads were washed with 1 liter
of extraction buffer followed by 500 ml of wash buffer (50 mM imidazole (pH 7), 150 mM NaCl, 5 mM sodium
-glycerophosphate, and 0.05% Triton X-100).
The washed agarose was poured into a column, and the M6P/IGF-II
receptor was eluted with wash buffer containing 10 mM
Man-6-P. Fractions containing the receptor were pooled, and protein
concentration was determined by the Bradford assay (47). The recovery
of the receptor was 660 µg.
Vo)/(Vt
Vo), was determined, and the
Erf
1 (1
Kd) was
plotted versus the known Stokes radius of the protein
standards (28). The Ve of the membrane form of the
M6P/IGF-II receptor was determined by collecting 1-ml fractions and
analyzing the contents by SDS-polyacrylamide gel electrophoresis
followed by Coomassie staining of the gel. The Ve of
complexes containing
-glucuronidase was determined by
-glucuronidase assays.
-glucuronidase assays as described below. The
sedimentation coefficients of the proteins and complexes were
determined using protein standards as markers (29).
-Glucuronidase Assays--
A sample of each fraction to be
tested (2-10 µl) was incubated with 100 µl of 10 mM
4-methylumbelliferyl
-D-glucuronide (Sigma) suspended in
0.1 M sodium acetate (pH 5.0) at 37 °C for 10-60 min.
Following incubation, 3 ml of 0.25 M glycine (pH 10.3) was added to stop the reaction, and the fluorescence was determined in a
Turner fluorometer.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Glucuronidase Are Internalized at Different
Rates--
The internalization rates of IGF-II and
-glucuronidase
were compared using 125I-IGF-II and
125I-
-glucuronidase in an adaptation of the endocytosis
assay developed by Jadot et al. (24). Following an initial
lag of 15-20 s, 125I-IGF-II was internalized in a nearly
linear fashion, with a t1/2 of 2-3 min (Fig.
1B). No plateau was observed
during the 5-min incubation because very little of the IGF-II was
released from the M6P/IGF-II receptor during the course of this assay.
The internalization of IGF-II occurred exclusively via the M6P/IGF-II
receptor since the untransfected parent cell line did not take up any
IGF-II under these conditions (data not shown).
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Fig. 1.
Internalization of IGF-II and
-glucuronidase occurs at different rates. Cells expressing the
wild-type receptor were incubated with 125I-labeled
ligand at 4 °C for 30 min, washed on ice to remove unbound ligand,
and then shifted to 37 °C for the indicated times. The amount of
ligand internalized is plotted as a fraction of the maximum possible
internalized counts, which is the sum of the internalized ligand plus
the ligand remaining on the cell surface at the end of the assay (see
"Experimental Procedures" for further explanation). Values are the
average of eight independent experiments, and the S.D. is indicated by
the error bars.
, internalized ligand (radioactivity that
is resistant to stripping with pH 3.5 for 10 min);
, surface ligand.
A, the ligand added was 125I-
-glucuronidase;
B, the ligand added was 125I-IGF-II.
-glucuronidase was internalized ~3-4-fold more
rapidly, with a t1/2 of 30-45 s (Fig.
1A). A plateau was reached when essentially all of the
ligand originally present on the cell had been either internalized or released from the receptor into the medium, where it was greatly diluted. Together, these data show that
-glucuronidase binding stimulates the rate of receptor internalization over that observed upon
IGF-II binding.
-Glucuronidase Stimulates Internalization of
125I-IGF-II, whereas Man-6-P Does Not--
To test whether
the increased rate of internalization of the receptor with bound
-glucuronidase was a result of ligand occupation of the two
Man-6-P-binding sites, the effect of 10 mM Man-6-P on the
rate of 125I-IGF-II uptake was determined. This
concentration of Man-6-P saturated the Man-6-P-binding sites on the
receptor. Although Man-6-P caused a small increase in total
125I-IGF-II binding, it had no effect on the rate of
125I-IGF-II internalization (data not shown), indicating
that the increase in internalization rate was not solely due to Man-6-P binding. The effect of
-glucuronidase on the internalization of
125I-IGF-II was next determined. In this experiment, the
simultaneous binding of 125I-IGF-II and
-glucuronidase
was maximized by first incubating cells on ice with
125I-IGF-II for 5 min to allow maximum binding of this
ligand. Excess unlabeled
-glucuronidase (10 nM) was then
added to each well for an additional 25 min on ice. The cells were
washed, and the uptake of 125I-IGF-II was determined. The
presence of
-glucuronidase stimulated the rate of endocytosis of
125I-IGF-II to that observed with
125I-
-glucuronidase alone (Fig.
2). This indicates that the unlabeled
-glucuronidase bound to a significant fraction of the receptors that
had already bound 125I-IGF-II, resulting in an increase in
internalization rate that cannot be merely due to the receptor binding
to Man-6-P residues.
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Fig. 2.
Unlabeled -glucuronidase enhances the rate
of internalization of 125I-IGF-II. Cells expressing
the wild-type receptor were assayed for internalization of
125I-
-glucuronidase (
), 125I-IGF-II in
the presence of 10 mM Man-6-P (
), or
125I-IGF-II in the presence of unlabeled
-glucuronidase
(
) as described under "Experimental Procedures." Values are the
average of three independent experiments.
-Glucuronidase could enhance the rate of internalization
of the M6P/IGF-II receptor by promoting either intramolecular or intermolecular cross-linking. Since each monomer of the receptor contains two Man-6-P-binding sites, simultaneous binding to two Man-6-P
residues on a phosphorylated oligosaccharide could induce a
conformational change in the extracellular domain of the receptor that
is transmitted to the cytosolic domain, where the internalization signal is located. This could result in a more favorable presentation of the internalization signal. Alternatively, the ligand could cross-link two receptor molecules, resulting in an increased density of
the internalization signals. This could enhance the likelihood of the
receptors being retained in a forming clathrin-coated pit, thus
increasing the probability of internalization and consequently the
rate. To distinguish between these possibilities, two approaches were
used. First, the effect of a small bivalent Man-6-P-containing peptide
on the rate of IGF-II uptake was determined. The peptide, a Thr-Lys-Thr
tripeptide with a Man-6-P(
1-2)Man disaccharide attached to each
threonine, has an affinity for the M6P/IGF-II receptor that is similar
to that of an oligosaccharide with two Man-6-P residues and over
1000-fold higher than that of Man-6-P (23, 30, 31). This high binding
affinity indicates that the ligand is interacting with two binding
sites on the M6P/IGF-II receptor. As shown below, this peptide does not
mediate intermolecular cross-linking of the receptor. In control
experiments, the peptide competed with
-glucuronidase for the
Man-6-P-binding site on the receptor, but did not interfere with
binding of IGF-II to the receptor (data not shown). A saturating
concentration of the peptide (5 µM) did not significantly
alter the rate of 125I-IGF-II internalization, whereas
unlabeled
-glucuronidase accelerated the rate of
125I-IGF-II uptake considerably (Fig.
3). These results suggest that intramolecular cross-linking of extracellular domains 3 and 9 of the
M6P/IGF-II receptor does not alter the rate of internalization.
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Fig. 3.
Intramolecular cross-linking does not enhance
internalization of 125I-IGF-II. The 344 cell line was
assayed for internalization of 125I-IGF-II in the presence
of 10 mM Man-6-P ( ), 5 µM tripeptide
containing two Man-6-P residues (
), or unlabeled
-glucuronidase
(
) as described under "Experimental Procedures." Values are the
average of three independent experiments. The 344 cell line expresses a
M6P/IGF-II receptor that has a 29-amino acid cytoplasmic tail ending in
the sequence AKYSKV. This receptor internalizes
-glucuronidase at
the same rate as the wild-type receptor, but expresses 4-fold more
receptor molecules at the cell surface, making it easier to perform the
assay (24).
) cells, creating the Dom3ala cell
line (25). The M6P/IGF-II receptors in this cell line are incapable of
intramolecular cross-linking due to the presence of only a single
functional Man-6-P-binding site per receptor, but could potentially
undergo intermolecular cross-linking.
-glucuronidase and IGF-II was determined (Fig.
4). In preliminary experiments,
internalization of 125I-IGF-II by Dom3ala cells
was partially obscured by a high background resulting from IGF-II
binding to other proteins (32). This technical problem was resolved by
using IGF-II-(del 1-6), which contains a deletion that prevents
binding to the IGF-II-binding proteins while maintaining internalization by the M6P/IGF-II receptor (32). IGF-II-(del 1-6) was
internalized by the wild-type M6P/IGF-II receptor at a rate similar to
that observed with native IGF-II (Fig. 4A), and the addition
of unlabeled
-glucuronidase increased the rate of receptor
internalization to that seen earlier (Fig. 2A). The rate of
125I-IGF-II-(del 1-6) uptake by the mutant receptor in the
Dom3ala cells was similar to that of the wild-type receptor
(Fig. 4B). This was expected since neither the
IGF-II-binding site nor the internalization signal in the cytoplasmic
domain is different. As with wild-type receptors, the addition of
unlabeled
-glucuronidase increased the rate of internalization of
the mutant receptor in Dom3ala cells, as measured by
125I-IGF-II-(del 1-6) uptake (Fig. 4B). This
strengthens the conclusion that intramolecular cross-linking is not
responsible for the increased rate of internalization and suggests that
intermolecular cross-linking mediates the enhanced internalization. The
internalization rate of 125I-IGF-II-(del 1-6) by the
Dom3ala cells in the presence of
-glucuronidase was not
as rapid as that seen with cells expressing the wild-type receptors.
This may be because the single Man-6-P-binding site on the mutant
receptor results in lower binding affinity for
-glucuronidase and
consequently less efficient cross-linking of receptor molecules.
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Fig. 4.
Intermolecular cross-linking is necessary for
stimulation of 125I-IGF-II internalization. The
rate of 125I-IGF-II-(del 1-6) internalization was
determined as described under "Experimental Procedures" in
the absence ( ) and presence (
) of unlabeled
-glucuronidase.
Values are the average of three independent experiments. The absence of
an error bar indicates that the S.D. was <0.02.
A, internalization by the wild-type M6P/IGF-II receptor;
B, internalization by the M6P/IGF-II receptor with a
mutation in domain 3 that abolishes its Man-6-P binding. Consequently,
this receptor can bind only one Man-6-P residue via domain 9.
-glucuronidase. Perdue
et al. (33) have reported that the M6P/IGF-II receptor is a
monomer when solubilized, whereas Stein et al. (34)
suggested that it may exist as a dimer in the plasma membrane, as
determined by cross-linking studies. The M6P/IGF-II receptor was
solubilized and purified from fresh bovine liver. The purified receptor
was analyzed by FPLC gel filtration to determine its Stokes radius (Fig. 5, A and C)
and by sedimentation in a continuous 6-21% sucrose gradient to
determine its sedimentation coefficient (Fig.
6). The Stokes radius of the receptor was
calculated to be 79 Å, which is somewhat greater than the previously
reported value of 72 Å (33). The sedimentation coefficient was
determined to be 10.1 × 1013 s, in close accordance
with the previously published value (33). The partial specific volume
was calculated to be 0.73, based on the amino acid composition and
expected carbohydrate additions. No corrections for detergent were
applied due to the negligible amount of bound detergent found by us
(compare migration relative to protein standards in H2O and
D2O gradients in Fig. 6) and others (33). Using these
values, the calculated molecular weight for the receptor was 334,000. This value is somewhat higher than previously published (290,000) due
to the slight difference in the Stokes radius. Nevertheless, the
calculated molecular weight indicates that the purified M6P/IGF-II
receptor solubilized in Triton X-100 is a monomer. Similar results were
obtained when the receptor was solubilized in digitonin (data not
shown).
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Fig. 5.
Determination of the Stokes radii of the
M6P/IGF-II receptor and the complex containing both the receptor and
-glucuronidase. A, 50 µg of the M6P/IGF-II receptor was
applied to a Superose 6 FPLC column (10 mm, inner diameter, × 28.5 cm), and 1-ml fractions were collected. The protein was precipitated
and analyzed by SDS-polyacrylamide gel electrophoresis followed by
Coomassie staining. The void volume and the elution positions of the
protein standards are indicated at the top. The M6P/IGF-II receptor
eluted in fraction 13. B, the M6P/IGF-II receptor was
incubated with
-glucuronidase (
-gluc) at a molar ratio
of 10 receptor molecules to 1 enzyme molecule for 30 min on ice. The
reaction mixture was then loaded onto the Superose 6 FPLC column, and
0.5-ml fractions were collected. Aliquots (10 µl) from each fraction
were analyzed for
-glucuronidase activity. The Ve
of free
-glucuronidase at 14.5 ml is indicated, as are the elution
positions of the protein standards. Complexed
-glucuronidase eluted
at 11.5 ml. C, the Stokes radii of the protein standards are
plotted against the inverse error coefficient of 1
Kd. These data were used to determine the Stokes
radii of the M6P/IGF-II receptor (X) and the complex
(Y). The protein standards and their Stokes radii are as
follows: B, thyroglobulin (Thy), 89 (41);
C, ferritin (Fer), 61 (42); D, BSA,
35.5 (42); E, ovalbumin, 27.5 (43); and F,
myoglobin, 18.9 (44). The void volume is indicated by A, and
the total volume of the column, as determined by the
Ve of cyanocobalamin, is indicated by
G.
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Fig. 6.
Determination of the sedimentation
coefficient of the M6P/IGF-II receptor. A and
C, purified M6P/IGF-II receptor (10 µg) was loaded onto a
continuous 6-21% sucrose gradient and centrifuged for 4 h at
50,000 rpm in an SW 55Ti rotor at 4 °C. Twenty fractions of 240 µl
were collected, and protein was precipitated and analyzed by
SDS-polyacrylamide gel electrophoresis followed by Coomassie staining.
Gradients were made in H2O (A) or
D2O (C). B and D, the
s20,w values of the protein standards
were plotted against the fraction they peaked in, and these data were
used to determine the s20,w of the
M6P/IGF-II receptor ( ). The marker gradients were run in
H2O (B) or D2O (D). The
protein standards were as follows: BSA,
s20,w = 4.6; lactate dehydrogenase
(LDH), s20,w = 7.3 (45);
catalase (Cat), s20,w = 11.3 (45); and thyroglobulin (Thy),
s20,w = 19.5 (46).
-glucuronidase on the oligomeric state of
the receptor, the ligand was incubated with the receptor for 30 min on
ice at a ratio of 10 receptor molecules to 1
-glucuronidase molecule. The resulting complex was then analyzed by gel filtration and
sucrose gradient sedimentation to allow calculation of the molecular
weight. The elution position of the receptor-
-glucuronidase complex
in the gel filtration column was determined by analyzing the fractions
for
-glucuronidase activity (Fig. 5B) and by Western blotting (data not shown). It is apparent that the complex eluted significantly ahead of free
-glucuronidase. Using this elution value, the Stokes radius was calculated to be 101 Å. The migration of
the M6P/IGF-II receptor-
-glucuronidase complex in the sucrose gradient was determined in a similar manner (Fig.
7A). The sedimentation value
of the complex was determined to be 21.6 × 1013 s
(Fig. 7B).
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Fig. 7.
Determination of the sedimentation
coefficient of the complex containing the M6P/IGF-II receptor and
-glucuronidase. A, the M6P/IGF-II receptor and
-glucuronidase were incubated together on ice for 30 min at a ratio
of 10 receptor molecules to 1 enzyme molecule and then loaded onto a
6-21% sucrose gradient made in H2O and centrifuged for
4 h at 50,000 rpm in an SW 55Ti rotor. Fractions (120 µl) were
collected and analyzed for
-glucuronidase activity. B,
the s20,w values of the protein
standards were plotted against the fraction they peaked in, and these
data were used to determine the s20,w of
the complex (
). The standard proteins are indicated at the top of
A as follows: A, BSA,
s20,w = 4.6; B, lactate
dehydrogenase (LDH), s20,w = 7.3; C, catalase, s20,w = 11.3; and D, thyroglobulin,
s20,w = 19.5.
-glucuronidase molecule, based on
the empirically determined values for the molecular weights of these
two proteins. Although the molecular weight of the complex could also
be consistent with the presence of one receptor and two
-glucuronidase molecules (expected molecular weight of 854,000),
this is unlikely because incubation of the proteins at a ratio of 10
-glucuronidase molecules to 1 receptor molecule gives rise to a
complex with a lower molecular weight, indicative of a complex of one
-glucuronidase molecule and one M6P/IGF-II receptor (25) (data not
shown). Thus, purified M6P/IGF-II receptor associates with
-glucuronidase in a complex composed of two receptors and a single
-glucuronidase molecule, consistent with the conclusion that
-glucuronidase can cross-link two M6P/IGF-II receptor molecules.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucuronidase three to four times more
quickly than the monovalent ligand IGF-II. This finding provides strong evidence that ligand binding can modulate the rate of trafficking of
the receptor, a point that has been open to debate in the literature (18-22). A key question is the mechanism of this effect. A clue came
from the fact that
-glucuronidase is a multivalent ligand with
multiple phosphorylated oligosaccharide units, whereas IGF-II is a
monovalent ligand. This raised the possibility that the
-glucuronidase effect arises from intermolecular cross-linking of
receptor molecules or by intramolecular cross-linking of the two
Man-6-P-binding sites located in domains 3 and 9 of the extracellular
domain. The latter possibility was excluded by the finding that the
Dom3ala mutant receptor with a single Man-6-P-binding site
retained the ability to respond to
-glucuronidase binding with an
increased rate of internalization, despite being incapable of
undergoing intramolecular cross-linking. The observation that a small
bivalent glycopeptide that binds to the receptor with high affinity
fails to enhance the rate of internalization is also consistent with this conclusion. This glycopeptide does not induce intermolecular cross-linking of receptor molecules due to its small size. On the other
hand, the in vitro binding studies with the purified receptor established that one molecule of
-glucuronidase cross-links two molecules of receptor, whereas IGF-II does not cross-link the receptor.
-glucuronidase increase the rate of internalization? One potential mechanism is that the cross-linking increases the efficiency of the
interaction between the tyrosine-based internalization signal present
in the cytoplasmic domain of the receptor and the AP-2 adaptor complex
at the site of clathrin-coated pit formation at the plasma membrane.
Fire et al. (35) have reported that productive interactions
with coated pits may be one of the rate-limiting steps for rapid
endocytosis of receptors. These investigators used fluorescence
photobleaching recovery measurements to determine the lateral diffusion
coefficient of wild-type influenza virus hemagglutinin, which is slowly
internalized, and a mutant hemagglutinin (Tyr-543) that is internalized
at a more rapid rate. Using these values and the size and number of
coated pits at the cell surface, they estimated that all the
hemagglutinin molecules encounter a coated pit every 3.7 s. They
concluded that the Tyr-543 mutant hemagglutinin enters and exits coated
pits many times before a productive interaction occurs since its
internalization rate is only 4%/min. In fact, most receptors judged to
undergo rapid endocytosis into clathrin-coated pits are internalized
with t1/2 values of 1 min or more, not seconds. This
implies either that their lateral diffusion is limited or that many of
the entries into coated pits fail to result in the trapping and
subsequent internalization of the protein. Collawn et al.
(36) found that adding a second internalization signal to the
cytoplasmic tail of the transferrin receptor increased its
internalization rate above that of the wild-type receptor. This
suggested that two internalization signals may be better than one.
Similarly, the cation-dependent Man-6-P receptor contains
three internalization signals, and its rate of internalization is
slowed when individual signals are mutated (37, 38).
-glucuronidase is added at saturation levels to cells kept at
4 °C, and the excess ligand is washed away before warming. Assuming
that the incubation on ice completely immobilizes receptors in the
plasma membrane, any intermolecular cross-linking of receptors must be
occurring in a rigid membrane. The implication is that the receptor
monomers are proximal to one another, perhaps already oligomerized,
before the addition of
-glucuronidase. If that is the case, the
enhanced internalization must be due to a more optimal presentation of
the internalization signal upon cross-linking with the multivalent ligand.
-glucuronidase, orienting the receptor with respect to the membrane
such that the Man-6-P-binding sites faced away from the membrane,
allowing docking of Man-6-P residues present on the oligosaccharides of
-glucuronidase. The large Stokes radius of the M6P/IGF-II receptor indicates that it is not globular in shape, but rather protrudes from
the membrane as a cigar-shaped molecule. Analysis of the oligomerization of the soluble receptor in association with
-glucuronidase revealed that binding of receptor molecules to this
multivalent ligand did not result in the formation of large multimers
despite the potential presence of up to 16 phosphorylated
oligosaccharides on the
-glucuronidase tetramer (40). Rather, a
discrete complex with a stoichiometry of two receptor molecules to one
enzyme formed, suggesting that steric hindrance may prevent more
receptors from binding. Taken together with the Stokes radius
information, this suggests a simple model for receptor-enzyme
association in which two M6P/IGF-II receptors cradle a single
-glucuronidase molecule between their extracellular domains,
covering most of the enzyme's surface. Functionally, this interaction
would prevent a single
-glucuronidase molecule from interacting with
M6P/IGF-II receptors on two cells, possibly preventing undesirable
intercellular adhesion from occurring.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA08759-30 (to S. K.), Postdoctoral Training Grant T32 HL07088 (to L. S. A.), and Grant DK42667 (to N. M. D.) and by Medical Scientist Training Program Training Grant T32 GM07200 (to S. J. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Medicine, Duke University Medical Center, Durham, NC 27706.
Performed this work during the tenure of an Established
Investigatorship from the American Heart Association.
** To whom correspondence should be addressed: Div. of Hematology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8125, St. Louis, MO 63110.
The abbreviations used are: M6P/IGF-II receptor, mannose 6-phosphate/insulin-like growth factor II receptor; IGF-II, insulin-like growth factor II; Man-6-P, mannose 6-phosphate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FPLC, fast protein liquid chromatography.
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