High-affinity ligand binding by wild-type/mutant
heteromeric complexes of the mannose
6-phosphate/insulin-like growth factor II receptor
Michelle A. Hartman
1
, Jodi L. Kreiling
2
, James C. Byrd
1
and Richard G. MacDonald
1
1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
2 Department of Chemistry, University of Nebraska, Omaha, NE, USA
The mannose 6-phosphate ⁄ insulin-like growth factor II
receptor (M6P ⁄ IGF2R) is a 300 kDa transmembrane
glycoprotein that has diverse ligand-binding properties
contributing to several important cellular functions
[1,2]. Insulin-like growth factor II (IGF-II) binding to
the M6P ⁄ IGF2R leads to uptake into the cell and deg-
radation of the growth factor in lysosomes [3–6]. This
activity reduces IGF-II availability in the pericellular
Keywords
insulin-like growth factor II; ligand binding;
mannose 6-phosphate; mannose
6-phosphate ⁄ insulin-like growth factor II
receptor; oligomerization
Correspondence
R. G. MacDonald, Department of
Biochemistry and Molecular Biology,
University of Nebraska Medical Center,
985870 Nebraska MED CTR, Omaha, NE

growth factor II receptor. Although wild-type ⁄ mutant hetero-oligomers
form readily when mixed, it appears that multivalent Man-6-P ligands bind
preferentially to wild-type sites, possibly by cross-bridging receptors within
clusters of immobilized receptors.
Abbreviations
Glc-6-P, glucose 6-phosphate; HA, hemagglutinin; HBS, Hepes-buffered saline; HBST, HBS containing 0.05% Triton X-100; IGF-II, insulin-like
growth factor II; M6P ⁄ IGF2R, mannose 6-phosphate ⁄ insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; pBSKII+,
pBluescript SK II+; PMP-BSA, pentamannosyl 6-phosphate-BSA.
FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1915
milieu, thereby decreasing its binding to mitogenic
IGF-I receptors, which contributes substantially to the
function of the M6P ⁄ IGF2R as a growth or tumor
suppressor. Binding of lysosomal enzymes by the
receptor is mediated by mannose 6-phosphate (Man-6-
P) groups on N-linked oligosaccharides, and this
mechanism is critical to lysosome biogenesis [7]. There
are also a number of glycoproteins other than the lyso-
somal enzymes that bind to the receptor by a Man-6-
P-dependent mechanism, including thyroglobulin,
proliferin, granzyme B and latent transforming growth
factor-b [1,2,8]. Several ligands, such as retinoic acid,
urokinase-type plasminogen activator receptor and
plasminogen, have also been reported to interact with
the M6P ⁄ IGF2R via novel binding sites [9–13].
The human M6P ⁄ IGF2R consists of a large extracy-
toplasmic domain (ectodomain) of 2265 amino acid
residues, a 23-residue transmembrane domain, and a
short, 164-residue cytoplasmic domain [14,15]. The
ectodomain comprises fifteen repeats having 14–28%
sequence identity. Each of the repeats is formed by a

dently of ligand binding, presumably mediated by
direct interactions between the ectodomains of each
monomer. Kreiling et al. [26] found that there is not
a specific M6P ⁄ IGF2R dimerization domain but,
rather, there are interactions that exist between dimer
partners all along the ectodomain of the receptor.
Collectively, these studies led to the hypothesis that
production of high-affinity ligand binding arises from
cooperation between Man-6-P binding sites on each
monomeric partner [1,27]. The dimer-based model for
high-affinity Man-6-P binding has recently received
support from structural analysis of repeats 1–3 of the
receptor’s ectodomain by Olson et al. [18]. Although
binding of IGF-II by the M6P ⁄ IGF2R does not
induce receptor dimerization [24] and it is known that
IGF-II binds the receptor with one-to-one stoichiome-
try [28], it remains unknown whether dimerization of
the receptor has any effect on IGF-II binding. That
is, would a defective IGF-II binding site on one
monomer interfere with IGF-II binding on the other
monomer?
The present study aimed to test the hypothesis
that IGF-II binds independently to its binding sites
on each receptor monomer, but that Man-6-P ligand
binding is bivalent, requiring cooperative interaction
of cognate sites on both monomers of the dimeric
receptor. To test this hypothesis, we measured ligand
binding by dimers formed from cDNA constructs
encoding repeats 1–15 of the M6P ⁄ IGF2R ectodo-
main. Our co-immunoprecipitation data indicate that

M6P/IGF2R mini-receptors
The M6P ⁄ IGF2R ectodomain is critical for receptor
ligand binding and dimerization [19,25,29,30]. There-
fore, receptor constructs for testing these functions
were designed to encode all 15 repeats of the ectodo-
main of the M6P⁄ IGF2R followed by either an eight-
residue FLAG epitope tag or a 12-residue Myc tag
(Fig. 1A). Distinct epitope tags were used to allow
detection of heterologous interactions between mini-
receptors. Two forms of the FLAG and Myc epitope-
tagged mini-receptors, 1-15 wild-type and 1-15 I1572T
(I ⁄ T), were transiently expressed alone or co-expressed
in HEK 293T human embryonic kidney cells. Cell
extracts were prepared using Triton X-100 and
analyzed for relative expression levels of the mini-
receptors by immunoblotting with M2 anti-FLAG or
9E10 anti-Myc immunoglobulins (Igs) (data not
shown).
The two differentially tagged mini-receptors were
constructed to assess the possibility of intersubunit
effects between receptors. Several studies have deter-
mined that the I1572T mutation residing in the heart
of the IGF-II binding domain in repeat 11 disrupts
IGF-II binding to the receptor [22,31–33]. The ligand
blotting data shown in Fig. 1C confirm that the wild-
type mini-receptors used in the present study could
bind IGF-II, whereas the I ⁄ T mutant mini-receptors
could not. By contrast, both wild-type and mutant
mini-receptors bound the phosphomannosylated
pseudoglycoprotein pentamannosyl 6-phosphate-BSA

tated mini-receptors was measured to assess if IGF-II
binds the wild-type mini-receptor in the presence of
the I ⁄ T mutant mini-receptors (Fig. 2B). It was pre-
dicted that the presence of the I ⁄ T mutant mini-recep-
tors would not interfere with IGF-II binding to the
wild-type receptors because IGF-II is a monovalent
ligand that should bind independently to each avail-
Myc
FLAG
FLAG
Myc
COOH
COOH
COOH
COOH
H
2
N
H
2
N
H
2
N
H
2
N
*
*
Construct name:

the carboxyl terminus, with repeats of the ectodomain shown as
rectangles. The shaded rectangles indicate repeats 3 and 9, to
which the main determinants of Man-6-P binding have been
mapped. The stippled rectangles represent repeat 11 containing
the principal residues responsible for IGF-II binding, and the aster-
isk denotes the I>T mutation at residue 1572 (I ⁄ T), which abro-
gates IGF-II binding. The black rectangles represent the FLAG or
Myc epitope tags on the carboxyl terminus. (B, C) Equimolar
amounts of the transfected cell lysates were electrophoresed on
6% nonreducing SDS ⁄ PAGE gels. The proteins were transferred to
BA85 nitrocellulose, processed for ligand blotting and probed for
binding of either [
125
I]PMP-BSA (B) or [
125
I]IGF-II (C) and developed
by autoradiography. The autoradiograms of representative blots are
shown.
M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R
FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1917
able receptor [24]. The data shown in Fig. 2B support
this idea because the total amount of IGF-II binding
tended to follow the line displayed in the bar graph,
which was calculated based on the percentage of wild-
type versus mutant receptor cDNAs input into the
original transfection (Fig. 2B).
Ligand binding by co-immunoprecipitated
FLAG- and Myc-tagged M6P/IGF2R mini-receptors
To determine whether co-transfected mini-receptors
interact in a possible oligomeric complex, 293T cell

and the other half formed heterodimers with the
FLAG-tagged mini-receptors. Figure 3C,D indicates
that the Myc-tagged mini-receptor did not immuno-
precipitate in the absence of a FLAG-tagged partner.
In addition, it is noteworthy that the presence of the
I ⁄ T mutation had no apparent effect on the interaction
leading to co-immunoprecipitation.
These data indicate that differentially epitope-tagged
M6P ⁄ IGF2R mini-receptors were capable of associa-
tion as asymmetric oligomers, but they do not indicate
whether these structures are functional in ligand bind-
ing. To test this property, co-immunoprecipitated
mini-receptors were subjected to direct binding analysis
using radiolabeled ligands (Fig. 3E,F). For this pur-
pose, differentially tagged mini-receptors were
co-immunoprecipitated using a FLAG-based antibody
from lysates of cells transfected with a 1 : 1 ratio of
receptor cDNAs. We would expect that approximately
25% of the Myc-tagged mini-receptors would be pres-
ent as homodimers, and thus would not precipitate in
this assay. Thus, it was projected that PMP-BSA bind-
ing to the co-immunoprecipitated mini-receptors would
yield approximately 75% of the binding observed with
individually immunoprecipitated FLAG-tagged mini-
0
30
30
µg 1-15F cDNA
µg 1-15I/T cDNA
0

125
I]IGF-II (B). The lines in each graph indicate the amount of
binding predicted if the wild-type and mutant receptors are binding
ligand independently. The triangles indicate a progressive shift in
the ratio of wild-type to mutant receptor cDNA transfected into
cells. Values represent the mean ± SD of three replicate measure-
ments for each condition. These data represent the means of four
independent experiments. [Correction added on 5 March 2009 after
first online publication: in Fig. 2B ‘
125
I-PMP-BSA binding’ was cor-
rected to ‘
125
I-IGF-II binding’.]
Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al.
1918 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS
receptors (Fig. 3E). Binding of [
125
I]PMP-BSA to
immunoprecipitated mini-receptors was not affected by
the proportion of wild-type versus I ⁄ T mutant mini-
receptors in the mixture, suggesting that the I ⁄ T muta-
tion did not interfere with the formation of oligomers
that are functional in phosphomannosyl ligand binding
and establishing a baseline of ligand binding function
(Fig. 3E).
Binding of [
125
I]IGF-II to immunoprecipitated mini-
receptors was measured to assess whether IGF-II binds

1-15F I/T
1-15Myc
1-15Myc I/T
30
0
0
0
15
15
0
0
15
15
0
0
0
0
15
15
15
15
0
0
0
0
0
30
30
0
0

1-15Myc
1-15Myc I/T
30
0
0
0
15
15
0
0
15
15
0
0
0
0
15
15
15
15
0
0
0
0
0
30
30
0
0
0

9E10 (C, D) Igs and developed with [
125
I]protein A. As a control,
cell lysate in the amount that was used during the immunoprecipi-
tation was directly loaded onto the gel (A, C). Cell lysates, contain-
ing equimolar amounts of expressed FLAG-tagged soluble
receptors, were immunoprecipitated with M2 anti-FLAG affinity
resin and then incubated in the presence of 1 n
M [
125
I]PMP-BSA (E)
or 2 n
M [
125
I]IGF-II (F) for 3 h at 4 °C. Bound ligand was determined
by centrifuging the resin pellets, washing and counting the pellets
in a c-counter. Radioactivity retained in the presence of either
5m
M Man-6-P or 1 lM IGF-II was subtracted from each binding
reaction to determine the specific binding for [
125
I]PMP-BSA and
[
125
I]IGF-II, respectively. The lines in each graph (E, F) indicate the
amount of binding predicted if the wild-type and mutant receptors
are binding ligand independently. The tables indicate the amounts
of the various cDNAs transfected into cells for each condition and
apply to the data shown both above and below the table. Values
represent the mean ± SD of three replicate measurements for

125
I]PMP-BSA
binding was calculated and represented according to
the line in the bar graph (Fig. 4E). The data shown in
Fig. 4E for the co-immunoprecipitated mini-receptors
were consistent with this expectation as well as the
results observed with complementary anti-FLAG
immunoprecipitation (Fig. 3E).
Binding of [
125
I]IGF-II to immunoprecipitated mini-
receptors was measured to determine whether IGF-II
binds independently to both sides of the asymmetric
hetero-oligomers (Fig. 4F). It was projected that the
percentage of binding would follow the line displayed
in the bar graph; however, when the I⁄ T mutant
Myc-tagged mini-receptor served as the bait for immu-
noprecipitation by the resin, binding of IGF-II to the
asymmetric hetero-oligomers was interfered with or
not detected as readily as expected. These results are
consistent with the results observed with anti-FLAG
immunoprecipitation from the same panel of mini-
receptor transfections (Fig. 3F). It appeared that, no
matter which epitope tag of the hetero-oligomer was
A
B
C
D
1-15F
1-15F I/T

E
F
1-15F
1-15F I/T
1-15Myc
1-15Myc I/T
30
0
0
0
15
15
0
0
15
15
0
0
0
0
15
15
15
15
0
0
0
0
0
30

α
-Myc
IP:
α
-Myc
IB:
α
-FLAG
IB:
α
-FLAG
IP:
α
-Myc
125
I-PMP-BSA binding
c.p.m. × 10
2
125
I-IGF-II binding
c.p.m. × 10
2
Fig. 4. Co-immunoprecipitation and ligand binding of FLAG and
Myc epitope-tagged asymmetric dimeric soluble receptors immuno-
precipitated with protein G-Sepharose. The ability of 1-15F to
co-immunoprecipitate with 1-15Myc was measured by immunopre-
cipitating equimolar amounts of the 1-15Myc soluble receptor in
293T lysates of the M6P ⁄ IGF2R mini-receptors with protein
G-Sepharose previously incubated with anti-Myc 9E10 Ig. After
immunoprecipitation, the resin pellets were collected, washed,

binding was suppressed when the tethering partner
(bait) was the I ⁄ T mutant. To test the possibility that
properties of the Myc epitope tag might somehow be
responsible for this phenomenon, the effects of a
different epitope tag, hemagglutinin (HA), were exam-
ined in pairing with FLAG-tagged receptors. However,
these results (data not shown) were consistent with
the results obtained with FLAG- and Myc-tagged
partners (Fig. 3F), even though a different epitope tag
(HA instead of Myc) was combined with the FLAG
epitope tag.
Ligand binding by double-mutant, FLAG-tagged
M6P/IGF2R mini-receptors
Two forms of the FLAG-tagged mini-receptors, 1-15
wild-type and 1-15 R426A ⁄ R1325A (R2A), were con-
structed to assess the possibility of intersubunit effects
for the phosphomannosyl ligand binding sites of the
mini-receptors (Fig. 5A). These mini-receptors were
transiently expressed alone or co-expressed in 293T
cells. Cell extracts were analyzed for relative expression
levels of the mini-receptors by immunoblotting with
M2 anti-FLAG Ig (data not shown).
Binding of [
125
I]IGF-II by immunoprecipitated
wild-type versus R2A mutant mini-receptors was inde-
pendent of the proportion of wild-type to mutant
mini-receptors in the mixture, suggesting that the R2A
mutation did not disrupt IGF-II binding (Fig. 5B).
These data support the hypothesis that IGF-II binding

COOH
COOH
*
1-15F R2A H
2
N
1-15F H
2
N
9
113
A
*
0
30
0
30
B
C
0.0
2.5
5.0
7.5
10.0
12.5
15.0
0
10
20
30

the amount of binding predicted if the wild-type and mutant recep-
tors are binding ligand independently. The triangles indicate a
progressive shift in the ratio of wild-type to mutant receptor cDNA
transfected into cells. Values represent the mean ± SD of three
replicate measurements for each condition. These data represent
the means of four independent experiments.
M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R
FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1921
be immunoprecipitated as oligomeric complexes. These
mini-receptors were transiently expressed alone or
co-expressed in 293T cells, and analyzed for relative
expression levels of the mini-receptors by immuno-
blotting with anti-FLAG and anti-Myc Igs (data not
shown).
The cell lysates with volumes normalized for expres-
sion of the FLAG-tagged mini-receptor were analyzed
by an immunoprecipitation assay using M2 anti-
FLAG affinity resin. PhosphorImager analysis of the
immunoblots confirmed that essentially all of the
expressed FLAG-tagged mini-receptors precipitated by
incubation with the M2 affinity resin (Fig. 6B versus
C). The data shown in Fig. 6D,E indicate that approx-
imately 50% of the co-expressed Myc-tagged
mini-receptors were co-immunoprecipitated with the
FLAG-tagged mini-receptors (Fig. 6D versus E),
suggesting a balanced distribution between Myc-tagged
mini-receptor homo-oligomers and hetero-oligomers
with the FLAG-tagged mini-receptors. The presence of
the R2A mutation had no detectable effect on the
interaction leading to co-immunoprecipitation.

15
15
30
0
0
0
0
0
30
0
0
0
0
30
Myc
Myc
COOH
COOH
HN
2
HN
2
*
1-15Myc
1-15Myc R2A
*
A
B
C
D

0
0
30
F
G
0
25
50
75
100
125
0
100
200
300
400
500
600
700
800
IB:
α
-FLAG
IB:
α
-FLAG
IP:
α
-FLAG
Transfected

shaded rectangles indicated repeats 3 and 9, to which the main
determinants of Man-6-P binding have been mapped and the aster-
isk denotes the RfiA mutations at residues 426 and 1325 (R2A),
which abrogates Man-6-P binding. The black rectangles represent
the Myc epitope tags on the carboxyl terminus. (B–E) The ability of
1-15Myc to co-immunoprecipitate with equimolar amounts of 1-15F
soluble receptor with M2 anti-FLAG affinity resin from 293T cell
lysates of M6P ⁄ IGF2R mini-receptors. After immunoprecipitation,
the resin pellets were collected, washed and immunoblotted with
anti-FLAG M2 (B, C) or anti-Myc 9E10 (C, D) Igs. As a control, cell
lysate in the amount that was used during the immunoprecipitation
was directly loaded on the gel (B, D). (F, G) Cell lysates, containing
equimolar amounts of expressed FLAG-tagged soluble receptors,
were immunoprecipitated with M2 anti-FLAG affinity resin and
assayed for binding of 2 n
M [
125
I]IGF-II (F) or 1 nM [
125
I]PMP-BSA
(G) for 3 h at 4 °C. The lines in each graph (F, G) indicate the
amount of binding predicted if the wild-type and mutant receptors
are binding ligand independently. The tables indicate the amounts
of the various cDNAs transfected into cells for each condition and
apply to the data shown both above and below the table. Values
represent the mean ± SD of three replicative measurements for
each condition. These data represent the means of four indepen-
dent experiments. [Correction added on 5 March 2009 after first
online publication: in Fig. 6E ‘IP: a -Myc’ was corrected to ‘IP:
a-FLAG’, and in Fig. 6F ‘

classes of ligands, IGF-II and phosphomannosylated
glycoproteins. In mammals, binding of IGF-II by the
M6P ⁄ IGF2R is thought to contribute to growth
homeostasis. Previous studies have shown that
the receptor operates optimally as a dimer and, in the
present study, we aimed to determine what effect the
dimeric structure may have on IGF-II binding. It has
been suggested that IGF-II binding to the
M6P ⁄ IGF2R requires contributions of repeats 11 and
13, but only within a single polypeptide chain
[20,22,33]. It is well established that the receptor binds
Man-6-P ligands in a multivalent fashion [1,18,35].
However, because the receptor has two Man-6-P bind-
ing domains within a single polypeptide chain, it
remains uncertain whether this bivalent binding activ-
ity is a property of a single monomeric receptor or the
result of cooperative interaction between the two
subunits of a dimeric receptor. There is strong evidence
that, in the cell, the preferred mode of binding is
through a dimeric structure, as shown by York et al.
[24], who found that a multivalent phosphomannosy-
lated ligand cross-bridged the dimeric receptor to pro-
mote optimal internalization. This conclusion was
reinforced by Byrd et al. [34], who analyzed mutant
receptors bearing a substitution of Arg for Ala at posi-
tion 1325 that knocks out Man-6-P ligand binding to
the repeat 9 site. Scatchard plot analysis showed that
these mutant receptors were still able to bind bivalent
Man-6-P ligands with high-affinity, leading to the con-
clusion that high-affinity binding in that case must be

epitope used to tag the receptor because there was no
discernable difference between the Myc- and
HA-tagged receptors in co-immunoprecipitation with
the FLAG-tagged receptor. As expected, we observed
that essentially all of the FLAG-tagged mini-receptors
were precipitated by incubation with M2 resin. In
experiments with the FLAG-tagged receptor as the
bait, approximately 50% of the Myc-tagged mini-
receptors were co-precipitated from a cell lysate pre-
pared from cells co-transfected with equal amounts of
the tagged mini-receptor cDNA. This strongly sug-
gests, but does not prove, that the mini-receptors asso-
ciated in a 1 : 2 : 1 relationship: 25% FLAG
homodimers, 50% FLAG-Myc heterodimers and 25%
Myc homodimers (which would not precipitate in this
assay). The simplest interpretation of these data rela-
tive to the structure of the receptor is that the mini-
receptors were in the form of dimers. Byrd et al. [34]
showed, via mutational analysis, that receptors with
only one functional Man-6-P binding site exhibited
high-affinity binding of Man-6-P-containing ligands.
Given that high-affinity binding of a bivalent ligand is
due to cooperative interaction with two or more recep-
tor binding sites [35], these data suggested that oligo-
merization of the receptor contributes to high-affinity
binding. In addition, native gel electrophoresis demon-
M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R
FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1923
strated that the receptor could be separated into
monomeric and dimeric forms in the presence or

containing extracytoplasmic repeats 10–13, which con-
tained the I1572T mutation in repeat 11. However, the
mechanism by which this mutation abrogates IGF-II
binding is still not clear. Structural analysis of repeat
11 identified the presumptive IGF-II binding site in a
hydrophobic pocket at the end of a b-barrel structure
[36]. Ile1572 was found to lie near, but not directly
within, this putative IGF-II binding site. This mutation
involves substituting a polar residue, Thr, for a bulky,
nonpolar residue, Ile, which might have altered the
IGF-II binding pocket by inducing a conformational
change that reduces binding energy or makes the site
less hydrophobic. In any case, this type of effect
should be regional and have minimal influence on the
wild-type IGF-II binding site on an adjacent mini-
receptor within a dimer. Our experiments support this
prediction, showing that the pairing of wild-type and
11572T mutant IGF-II binding sites between two
dimerized mini-receptors had no effect on the function
of the contralateral binding site. The mutant site does
not prevent the wild-type site from binding IGF-II and
pairing with a wild-type subunit does not repair the
defect in the mutant site inducing it to bind IGF-II.
This indicates that IGF-II binding to each side of the
dimer is independent. Symmetric heterodimers (i.e.
having identical epitope tags, both of which are teth-
ered to the resin bead) achieve the predicted amount
of binding as described above. Tethering of both sides
of the dimer likely mimics the structure obtained when
anchored in the membrane, in accordance with the

plasmic domain results in the inability to form appro-
priate contacts between dimeric partners. Follow-up
experiments using structural approaches are required
to address this possibility.
Localization of the two Man-6-P binding domains
was previously reported by Westlund et al. [30], who
subjected the M6P ⁄ IGF2R to partial proteolytic diges-
tion using subtilisin. They determined that repeats 1–3
and 7–10 can independently bind Man-6-P-containing
ligands. Dahms et al. [19] further defined the location
of Man-6-P binding sites by using mutational analysis
to establish the importance of specific Arg residues in
the function of both Man-6-P binding sites. They
determined that Arg426 and Arg1325 in repeats 3 and
9, respectively, are essential components of the recep-
tor’s high-affinity Man-6-P binding sites. The structure
of repeats 1–3 of the bovine M6P ⁄ IGF2R in the pres-
ence of Man-6-P was solved by Olson et al. [18]. Their
Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al.
1924 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS
work revealed key amino acid residues in the binding
site of repeat 3 that were important for Man-6-P bind-
ing. In particular, it was found that the guanidinium
group of Arg435 (corresponding to Arg111 of the cat-
ion-dependent mannose 6-phosphate receptor and
Arg426 of the human M6P ⁄ IGF2R) forms one or two
critical hydrogen bonds with the 2-hydroxyl group of
the mannose ring but these represent only two out of
more than a dozen noncovalent interactions between
ligand and receptor within the binding pocket. This

trated in coated pits on the plasma membrane and that
there is a > 60-fold enrichment of receptor in clathrin-
coated pits compared to microsomes [37,38]. During
receptor-mediated endocytosis, when there is a high
accumulation of receptors into one location, it is possi-
ble they can interact in higher-order oligomeric struc-
tures. This would explain why we do not observe a
decrease in affinity of phosphomannosyl ligands
between wild-type and R2A mutant receptors. This
accumulation of receptors may be mimicked on the
resin bead during an immunoprecipitation such that
the multivalent ligand binding selects binding-compe-
tent partners, promoting preferential cross-bridging
between wild-type receptors and forming higher oligo-
meric structures on the resin bead. As observed with
the IGF-II binding mutant, asymmetric heterodimers
that are tethered by mutant receptors show an
unexpected decrease in PMP-BSA binding. It thus
appears that tethering of the C-terminal end of the
ectodomain is important for both IGF-II and PMP-
BSA binding.
In summary, the major findings obtained in the
present study are consistent with a dimer model for
M6P ⁄ IGF2R oligomerization because all co-immuno-
precipitations resulted in the predicted outcome of
pull-down or ligand binding irrespective of the tag or
mutant. IGF-II was found to bind independently to
sites on each monomeric partner, whereas high-affinity
binding of multivalent Man-6-P ligands was propor-
tional to the number of wild-type binding sites avail-

I] and
[
125
I]protein A were obtained from PerkinElmer Life Sci-
ences (Boston, MA, USA). Recombinant human IGF-II
was a gift of Lilly Research Laboratories (Indianapolis,
IN, USA). The pseudoglycoprotein PMP-BSA was pre-
pared as described previously [34]. Radiolabeled PMP-
BSA was prepared by iodination using precoated IODO-
GEN tubes from Pierce (Rockford, IL, USA) according to
M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R
FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1925
the manufacturer’s instructions to a specific activity of 25–
70 CiÆg
)1
. IGF-II was radioiodinated using Enzymobeads
from Bio-Rad (Hercules, CA, USA) to a specific activity
of 33–114 CiÆg
)1
. The pCMV5 vector was provided by
David W. Russell (University of Texas Southwestern Med-
ical Center, Dallas, TX, USA) [39]. The 8.6 kb pair
human M6P ⁄ IGF2R cDNA was a gift of William S. Sly
(St Louis University Medical Center, St Louis, MO, USA)
[14]. Oligonucleotides were synthesized by Integrated
DNA Technologies (Coralville, IA, USA). All other
reagents and supplies were obtained from sources as indi-
cated.
Preparation of epitope-tagged soluble receptors
A soluble receptor containing all 15 extracytoplasmic

sequence corresponding to nucleotides 1112–1129 of the
receptor cDNA and a 3¢-primer with sequence complemen-
tary to nucleotides 2474–2487 of M6P ⁄ IGF2R cDNA,
followed by an XhoI site. The products from these
amplifications were digested with EcoRI and Xho I and sub-
cloned into pBluescript SK II+ (pBSKII+) (Stratagene).
This construct was subjected to two rounds of amplification
with primers designed to incorporate the R426A mutation
responsible for altering the ectodomain repeat 3 Man-6-P
binding site using the Megaprimer approach [41]. This first
round of amplification involved producing the mutation, by
amplifying from that site (nucleotide 1425) to the 3¢-end of
the mini-receptor (nucleotide 2487). This ‘megaprimer’ was
then used in a second round of amplification with the
5¢-primer used previously. The repeat 3 mutant amplifica-
tion product was digested with EcoRI and XhoI and subcl-
oned back into pBSKII+. An approximately 1 kb BsmI
fragment (sites at M6P ⁄ IGF2R nucleotides 1408 and 2449)
containing the mutation was removed from the megaprimer
and subcloned into the corresponding positions of
pBSKII+ ⁄ Kpn, which contained a 2.2 kb KpnI fragment
derived from M6P ⁄ IGF2R nucleotides 100–3242, creating a
pBSKII+ ⁄ Kpn-R426A mini-receptor with the third repeat
Man-6-P-binding mutation. The KpnI fragment from this
construct was then subcloned into pCMV5 ⁄ R1325A that
encoded either a FLAG- or Myc-tagged soluble receptor
containing the ninth repeat Man-6-P-binding mutation
synthesized as described previously [34], which had also
been digested with KpnI and XmnI, creating a soluble
M6P ⁄ IGF2R mini-receptor bearing Man-6-P binding-site

of autoradiography followed by PhosphorImager (Molecu-
lar Dynamics, Sunnyvale, CA, USA) to quantify relative
expression of the soluble receptors.
Ligand blot analysis
Aliquots of 293T cell lysates, containing equimolar
amounts of expressed soluble receptors, were electrophore-
sed on a 6% SDS ⁄ PAGE gel under nonreducing conditions
Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al.
1926 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS
and transferred to BA85 nitrocellulose paper. The ligand
blots were processed according to the procedure of
Hossenlopp et al. [43], probed using [
125
I]IGF-II ( 2 ·
10
6
c.p.m. per 8 mL) or [
125
I]PMP-BSA ( 2 · 10
6
c.p.m.
per 8 mL) for 16 h at 4 °C, and then developed by auto-
radiography.
Co-immunoprecipitation of soluble receptors
with M2 anti-FLAG affinity resin
Aliquots of 293T cell lysates, containing equimolar
amounts of expressed FLAG-tagged soluble receptors, were
incubated with 8 lL of packed M2 affinity resin in Hepes-
buffered saline (HBS; 50 mm Hepes, pH 7.4, 0.15 m NaCl)
plus 1% BSA and 5 mm Man-6-P for 3 h at 4 °Conan

125
I]protein A, followed by autoradiography and Phos-
phorImager analysis.
[
125
I]IGF-II and [
125
I]PMP-BSA binding analysis
Aliquots of 293T cell lysates containing equimolar amounts
of the expressed FLAG-tagged receptors were incubated
with 8 lL of packed M2 affinity resin in HBS + 1% BSA
for 3 h at 4 °C. The addition of 5 mm Man-6-P at this
point prevented the co-precipitation of endogenous phos-
phomannosylated ligands. The resin was collected by centri-
fugation at 13 000 g for 20 s. The resin pellets were washed
three times with 1.0 mL of HBST. The ability of the immu-
noprecipitated receptors to bind [
125
I]IGF-II was measured
by incubating the resin pellets with 2 nm [
125
I]IGF-II plus
100 nm unlabeled IGF-I in HBST for 16 h at 4 °C. The
addition of IGF-I to the binding reaction prevented inter-
ference from IGF-binding proteins that exist in the cell
lysates. The resin pellets were washed twice with 1.0 mL of
HBST to remove unbound ligand, collected by centrifuga-
tion, and counted in a c-counter. Specific [
125
I]IGF-II bind-

ter Monoclonal Antibody Facility for the production
of the 9E10 antibody. We also thank Christine
D. Dreis, Rosslyn Grosely and Christopher M. Con-
nelly for their input and technical support. This work
is supported in part by National Institutes of Health
Grant 5RO1CA91885 to R.G.M. J. L. Kreiling was
the recipient of pre-doctoral stipend support provided
by Graduate Studies; Bukey, McDonald, Emley and
Widaman fellowships; the Dr Fred W. Upson Grant-
in-aid award through the University of Nebraska
Medical Center and the NASA Space Grant
Fellowship. M. A. Hartman was the recipient of pre-
doctoral stipend support provided by Graduate
Studies and Skala Fellowships through the University
of Nebraska Medical Center and the GAANN
Fellowship.
M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R
FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1927
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