Tài liệu Báo cáo khoa học: High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose pptx

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

68198 5870, USA
Fax: +1 402 559 6650
Tel:+1 402 559 7824
E-mail:
(Received 17 October 2008, revised 19
December 2008, accepted 21 January 2009)
doi:10.1111/j.1742-4658.2009.06917.x
The mannose 6-phosphate ⁄ insulin-like growth factor II receptor has diverse
ligand-binding properties contributing to its roles in lysosome biogenesis
and growth suppression. Optimal receptor binding and internalization of
mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric struc-
ture leading to bivalent high-affinity binding, presumably mediated by
cooperation between sites on both subunits. Insulin-like growth factor II
(IGF-II) binds to a single site on each monomer. It is hypothesized that
IGF-II binding to cognate sites on each monomer occurs independently,
but bivalent Man-6-P ligand binding requires cooperative contributions
from sites on both monomers. To test this hypothesis, we co-immunopre-
cipitated differentially epitope-tagged soluble mini-receptors and assessed
ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites
between two dimerized mini-receptors had no effect on the function of the
contralateral binding site, indicating IGF-II binding to each side of the
dimer is independent and manifests no intersubunit effects. As expected,
heterodimeric receptors composed of a wild-type monomer and a mutant
bearing two Man-6-P-binding knockout mutations form functional IGF-II
binding sites. By contrast to prediction, such heterodimeric receptors also
bind Man-6-P-based ligands with high affinity, and the amount of binding
can be attributed entirely to the immunoprecipitated wild-type receptors.
Anchoring of both C-terminal ends of the heterodimer produces optimal
binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds indepen-
dently to both subunits of the dimeric mannose 6-phosphate ⁄ insulin-like

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

disulfide-bonded, crossed antiparallel b-sheet sandwich
that resembles a flattened b-barrel [16]. Ligand binding
experiments have mapped the Man-6-P binding
domains mainly to repeats 3 and 9, wherein mutation
of critical residues can reduce ligand affinity [17], and
such mapping is in agreement with the structure of
repeat 3 deduced from X-ray crystallography [18]. The
main amino acid residues involved in IGF-II binding
are located within repeat 11, but residues within repeat
13 cooperate with repeat 11 to enhance ligand binding
affinity [19–22].
Until recently, the M6P ⁄ IGF2R was considered to
be monomeric in structure, and this view was sup-
ported by studies on the physicochemical properties
of the solubilized receptor [23]. However, a recent
study by York et al. [24] demonstrated that phos-
phomannosyl ligands with multiple Man-6-P moieties,
such as b-glucuronidase, induced the cell-surface
M6P ⁄ IGF2R to form dimers, which enhanced the
rate of receptor internalization. These studies
provided the first evidence of ligand-mediated cross-
bridging of receptor monomers into a dimeric struc-
ture that interacted with apparently increased
efficiency with the endocytic apparatus. IGF-II bind-
ing failed to produce such an increase in receptor
internalization, which supported the hypothesis that
IGF-II binds to its sites on the individual monomeric
receptors [24]. Further insight into the mechanism of
dimerization was provided by Byrd et al. [25], who
showed that dimer formation could occur indepen-

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

oligomer formation does occur between receptors
bearing different C-terminal epitope tags. Hetero-
dimeric receptors composed of a wild-type monomer
and a mutant bearing an IGF-II binding knockout
mutation can form fully functional phosphomannosyl
binding sites. By contrast, such receptor dimers are
capable of binding IGF-II to the wild-type side, but
not to the mutant side of the dimer. Overall, the
analysis of IGF-II binding in such receptor dimers
suggests that each half of the dimer is capable of
binding IGF-II independently of the ligand
occupancy of the contralateral site. A heterodimeric
receptor composed of a wild-type monomer and a
mutant bearing two Man-6-P binding knockout
mutations can form functional IGF-II binding sites.
However, such receptors are also capable of
high-affinity Man-6-P binding, with the amount of
ligand binding being directly proportional to the
amount of the wild-type receptor present. These
results can be explained either by a sterically
improbable intramolecular binding mechanism or by
binding of a multivalent ligand forcing receptors to
realign within the immunoprecipitated complexes,
thus promoting preferential cross-bridging between
wild-type receptors.
Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al.
1916 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS
Results
Transient expression and ligand binding
properties of FLAG and Myc epitope-tagged

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

(PMP-BSA) (Fig. 1B). The presence of the different
epitope tags on the mini-receptors had no apparent
effect on ligand binding in this assay.
Ligand binding by immunoprecipitated
FLAG-tagged M6P/IGF2R mini-receptors
Previous studies suggested the possibility of negative
cooperativity of ligand binding by the oligomeric
M6P ⁄ IGF2R [34]. Thus, prior to examination of
FLAG- and Myc-tagged mini-receptors, we assessed
the ligand binding characteristics of a mixture of wild-
type and mutant FLAG-tagged mini-receptors. To
accomplish this, cells were transfected with a mixture
of cDNAs in which the proportion of mutant cDNA
to wild-type cDNA was increased, whereas the total
amount of cDNA was held constant. The effects of the
I ⁄ T mutation on both IGF-II and PMP-BSA binding
were analyzed using FLAG-tagged mini-receptors in a
mixed immunoprecipitation, which ensured that both
C-terminal ends were anchored to the resin in the same
way (Fig. 2). Binding of [
125
I]PMP-BSA to mixed
mini-receptors, which was measured to establish a
baseline of ligand binding function, was not affected
by the proportion of wild-type to mutant mini-recep-
tor, suggesting that the I ⁄ T mutation did not interfere
with functional phosphomannosyl ligand binding
(Fig. 2A). Binding of [
125
I]IGF-II to immunoprecipi-

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:

1-15F
1-15F I/T
1-15Myc
1-15Myc I/T
9
113
51
-
1
F
1-15F I/T
-
151
My
c
1-15
M
cIT
y
/
IGF-II
PMP-BSA
Endogenous
Endogenous
A
B
C
Fig. 1. Schematic diagram and ligand blot analysis of FLAG and
Myc epitope-tagged M6P ⁄ IGF2R mini-receptors. (A) The receptor
constructs are shown in linear format from the amino terminus to

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

lysates containing co-expressed FLAG and Myc
epitope-tagged mini-receptors were analyzed by an
immunoprecipitation assay using M2 anti-FLAG
affinity resin. The resin pellets were washed to remove
unbound proteins, separated by reducing SDS ⁄ PAGE,
and analyzed by immunoblotting with anti-FLAG or
anti-Myc Igs to determine whether the Myc epitope-
tagged mini-receptors interacted with the FLAG
epitope-tagged mini-receptors (Fig. 3A–D). Cells were
transfected either with 30 lg of cDNA encoding the
FLAG- and Myc-tagged mini-receptors alone or with
a combination of 15 lg of each differentially tagged
mini-receptor cDNA. PhosphorImager analysis of the
blots revealed that essentially all of the expressed
FLAG-tagged mini-receptors precipitated by incuba-
tion with the M2 affinity resin (Fig. 3A versus B). If
the level of expression of mutant versus wild-type
receptors reflects the proportion of their respective
cDNAs in the transfection, and based on random asso-
ciation to form dimers, it was projected that co-immu-
noprecipitation of the FLAG-tagged mini-receptors
from a 1 : 1 transfection pool would yield a 1 : 2 : 1
distribution of wild-type homodimers, wild-type ⁄
mutant heterodimers and mutant homodimers, respec-
tively. Figure 3C,D indicates that approximately 50%
of the co-expressed Myc-tagged mini-receptors were
co-immunoprecipitated with the FLAG-tagged mini-
receptors (Fig. 3C versus D), suggesting that approxi-
mately half the Myc-tagged mini-receptors existed as
homodimers (which do not precipitate in this assay)

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

0
25
50
75
100
125
150
125
I-PMP-BSA binding
c.p.m. × 10
3
125
I-IGF-II binding
c.p.m. × 10
3
0
10
20
30
40
A
B
Fig. 2. Analysis of ligand binding to soluble 1-15 and 1-15 I ⁄ T
mutant FLAG epitope-tagged receptors immunoprecipitated with
anti-FLAG resin. Cell lysates, containing equimolar amounts of
expressed soluble receptors, were immunoprecipitated with M2
anti-FLAG affinity resin and assayed for binding of [
125
I]PMP-BSA
(A) or [

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

independently to the asymmetric heterodimers
(Fig. 3F). In this assay, we expected that 25% of the
dimers formed would be Myc-tagged symmetric
homodimers and therefore would not be immuno-
precipitated by the M2 resin, 25% would be FLAG-
tagged symmetric homodimers, and the remaining
50% of the dimers formed would be FLAG- and
Myc-tagged asymmetric heterodimers. The calculations
projected that the percentage of binding would follow
the line displayed in the bar graph. However, the data
show that when a mutant FLAG-tagged mini-receptor
served as the ‘bait’ for immunoprecipitation, binding
of IGF-II to the asymmetric heterodimers was sup-
pressed or not detected as readily as expected. One
possibility for explaining this functional deficit may
comprise interference from the pairing of two different
C-terminal epitope tags.
Ligand binding by FLAG- and Myc-tagged
M6P/IGF2R mini-receptors via reciprocal
co-immunoprecipitation
To test whether the FLAG or Myc epitope tags inter-
fere with the formation of fully functional receptors in
asymmetric heterodimers by having only half of the
complex tethered to the resin, a reciprocal immunopre-
cipitation was performed. Cell lysates containing
co-expressed FLAG and Myc epitope-tagged mini-
Transfected
construct
(µg)
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
30
0
0

0
IB: α
α
-FLAG
IB:
α
-FLAG
IB:
α
-Myc
IB:
α
-Myc
IP:
α
-FLAG
IP:
α
-FLAG
C
D
E
F
B
A
Transfected
construct
(µg)
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
30
0
0
0

0
25
50
75
100
125
150
125
I-PMP-BSA binding
c.p.m. × 10
2
0
10
20
30
40
125
I-IGF-II binding
c.p.m. × 10
3
Fig. 3. Co-immunoprecipitation and ligand binding by FLAG and
Myc epitope-tagged asymmetric dimeric soluble receptors immuno-
precipitated with M2 anti-FLAG resin. The ability of 1-15Myc to
co-immunoprecipitate with 1-15F was measured by immunoprecipi-
tating equimolar amounts of the 1-15F soluble receptor with M2
anti-FLAG affinity resin from 293T lysates of the M6P ⁄ IGF2R
mini-receptors. After immunoprecipitation, the resin pellets were
collected, washed, heated with sample buffer and analyzed by 6%
reducing SDS ⁄ PAGE. The proteins were transferred to BA85 nitro-
cellulose, immunoblotted with anti-FLAG M2 (A, B) or anti-Myc

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

each condition. These data represent the means of four indepen-
dent experiments. [Correction added on 5 March 2009 after first
online publication: in Fig. 3D ‘IP: a-Myc’ was corrected to ‘IP:
a-FLAG’, and in Fig. 3F ‘
125
I-PMP-BSA binding’ was corrected to
‘
125
I-IGF-II binding’.]
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 1919
receptors were analyzed by an immunoprecipitation
assay using protein G-Sepharose and 9E10 anti-Myc
Ig. Immunoblots revealed that essentially all of the
input Myc-tagged mini-receptors precipitated in the
assay (Fig. 4A versus B). Figure 4C,D indicates that
approximately 50% of the co-expressed FLAG-tagged
mini-receptors were co-immunoprecipitated with the
Myc-tagged mini-receptors (Fig. 4C versus D). As was
observed in anti-FLAG-based immunoprecipitations
(Fig. 3), the presence of the I ⁄ T mutation had no
effect on the interaction leading to co-immunoprecipi-
tation.
To assess the ligand binding function of these asym-
metric heterodimers, co-immunoprecipitated mini-
receptors were subjected to direct binding analysis
using radiolabeled ligands (Fig. 4E,F). Based on the
premise that only 75% of the dimers formed during
this assay would be precipitable using the Myc-based
immunoprecipitation, the amount of [

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

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

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

30
0
0
0
0
10
20
30
40
50
0
10
20
30
40
50
60
70
80
90
Transfected
construct
(µg)
Transfected
construct
(µg)
IB:
α
-Myc
IB:

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,

heated with sample buffer and analyzed by 6% reducing
SDS ⁄ PAGE. The proteins were transferred to BA85 nitrocellulose
and immunoblotted with anti-Myc 9E10 (A, B) or anti-FLAG M2 (C,
D) Igs. As a control, cell lysate in the amount that was used during
the immunoprecipitation was directly loaded onto the gel (A, C).
Cell lysates, containing equimolar amounts of expressed
Myc-tagged soluble receptors, were immunoprecipitated with pro-
tein G-Sepharose previously incubated with anti-Myc 9E10 Ig and
assayed for binding of [
125
I]PMP-BSA (E) or [
125
I]IGF-II (F). 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 representative measurements for each condition. These data
represent the means of four independent experiments. [Correction
added on 5 March 2009 after first online publication: in Fig. 4D ‘IP:
a-FLAG’ was corrected to ‘IP: a-Myc’, and in Fig. 4F ‘
125
I-PMP-BSA
binding’ was corrected to ‘
125
I-IGF-II binding’.]
Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al.
1920 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS
anchored to the immunoprecipitating resin, IGF-II

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

to the co-immunoprecipitated mini-receptors would be
almost the same as that observed with the mini-recep-
tors expressed individually. Binding of [
125
I]PMP-BSA
to immunoprecipitated mini-receptors was measured to
assess whether the presence of the mutant mini-recep-
tors affected PMP-BSA binding to the wild-type mini-
receptors (Fig. 5C). In these experiments, the null
hypothesis proposes that there is no cross-talk between
binding sites within the mixture and thus binding
should simply reflect the proportions of wild-type and
mutant receptors in the transfection panel. Thus, we
projected that the percentage of binding based on
contributions of the mutant (no binding activity) and
wild-type (100% binding activity) mini-receptors in the
mixture would follow the line displayed in the bar
graph, suggesting that the wild-type binding site on one
receptor is not affected by the presence of a mutant
mini-receptor that is incapable of binding PMP-BSA.
Ligand binding by double-mutant FLAG and
Myc-tagged co-immunoprecipitated M6P/IGF2R
mini-receptors
Two forms of the Myc epitope-tagged mini-receptors,
1-15 wild-type and 1-15 R426A ⁄ R1325A (R2A)
(Fig. 6A), were constructed to assess whether these
co-transfected differentially tagged mini-receptors can
Construct name:
FLAG
FLAG

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

40
125
I-IGF-II binding
c.p.m. × 10
2
125
I-PMP-BSA binding
c.p.m. × 10
3
µg 1-15F cDNA
µg 1-15F R2A cDNA
Fig. 5. Schematic diagram and ligand binding analysis of soluble
1-15 and 1-15R2A mutant FLAG epitope-tagged receptors immuno-
precipitated with anti-FLAG resin. (A) The receptor constructs are
shown in linear format from the amino terminus to the carboxyl
terminus, with repeats of the ectodomain illustrated as rectangles.
The stippled rectangles represent repeat 11 containing the principal
residues responsible for IGF-II binding. The shaded rectangles indi-
cated repeats 3 and 9, to which the main determinants of Man-6-P
binding have been mapped and the asterisk denotes the RfiA
mutations at residues 426 and 1325 (R2A), which abrogates Man-
6-P binding. The black rectangles represent the FLAG epitope tags
on the carboxyl terminus. (B, C) Cell lysates, containing equimolar
amounts of expressed soluble receptors, were immunoprecipitated
with M2 anti-FLAG affinity resin and assayed for binding of
[
125
I]IGF-II (B) or [
125
I]PMP-BSA (C). The lines in each graph indicate

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.

Binding of [
125
I]IGF-II to immunoprecipitated wild-
type versus R2A mutant mini-receptors was equivalent,
suggesting that the R2A mutation had no discernible
effect on the formation of oligomers that are func-
tional in IGF-II binding (Fig. 6F). These data were
consistent with the prediction that, because 25% of the
oligomers formed should be homo-oligomers of Myc-
tagged mini-receptors, IGF-II binding by the co-immu-
noprecipitated mini-receptors should have yielded
approximately 75% of the binding observed with
1-15F
1-15F R2A
1-15Myc
1-15Myc R2A
30
0
0
0
15
15
0
0
15
15
0
0
0
0

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

E
1-15F
1-15F R2A
1-15Myc
1-15Myc R2A
30
0
0
0
15
15
0
0
15
15
0
0
0
0
15
15
30
0
0
0
0
0
30
0
0

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

construct
(µg)
Transfected
construct
(µg)
IB:
α
-Myc
IB:
α
-Myc
IP:
α
-FLAG
125
I-IGF-II binding
c.p.m. × 10
2
125
I-PMP-BSA binding
c.p.m. × 10
3
Fig. 6. Schematic diagram of soluble 1-15 and 1-15 R2A mutant
Myc epitope-tagged receptors, co-immunoprecipitation and ligand
binding analysis of soluble 1-15 and 1-15R2A mutant FLAG and
Myc epitope-tagged asymmetric soluble heterodimeric receptors.
(A) The receptor constructs are shown in linear format from amino
terminus to carboxyl terminus, with repeats of the ectodomain illus-
trated as rectangles. The stippled rectangles represent repeat 11
containing the principal residues responsible for IGF-II binding. The

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 ‘

125
I-PMP-BSA binding’ was corrected to
‘
125
I-IGF-II binding’.]
Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al.
1922 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS
individually immunoprecipitated, FLAG-tagged mini-
receptors.
Binding of [
125
I]PMP-BSA to these asymmetric het-
ero-oligomers was measured to determine whether
PMP-BSA binding would show interactive effects
between the wild-type and mutant partners (Fig. 6G).
As observed previously when a mutant FLAG-tagged
mini-receptor acted as the bait molecule in the immu-
noprecipitation, binding of PMP-BSA to the asym-
metric hetero-oligomers was suppressed. These results
are consistent with the results obtained in experiments
with the I ⁄ T mutation, suggesting that the ligand bind-
ing functions of the receptor’s ectodomain can operate
independently of one other within each receptor and
relative to receptor partners, again indicating that this
effect depends on tethering the C-terminal ends of the
extracytoplasmic domains.
Discussion
The rationale for the present study was to improve
understanding of how the subunits of the multimeric
M6P ⁄ IGF2R participated in binding the two main

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

due to alignment of two repeat 3 Man-6-P binding
domains on paired monomers. Furthermore, Olson
et al. [16] demonstrated, via X-ray projection models,
that the closest distance between the two Man-6-P
binding sites of one monomeric receptor is 45–70 A
˚
,
indicating that a single diphosphorylated oligosaccha-
ride, with a maximum distance of approximately 30 A
˚
[35], could not bind to the Man-6-P binding domains
of both repeats 3 and 9 simultaneously [16]. The pres-
ent study was designed to test whether ligand binding
by the dimeric receptor is cooperative, based on the
hypothesis that IGF-II binds independently to cognate
sites on both monomers of the dimeric receptor, but
that Man-6-P ligand binding would require coopera-
tion of both monomers. For this purpose, we devel-
oped a quantitative assay for heterodimer formation
that was based on immunoprecipitation of differen-
tially epitope-tagged receptors. The availability of the
I1572T mutant allowed us to address the initial
question of whether a nonfunctional IGF-II binding
site would interfere with the function of a wild-type
binding site when paired in a single heterodimeric
structure.
Association assays indicated that immunoprecipita-
tion between differentially epitope-tagged mini-recep-
tors was feasible. These assays also indicated that
immunoprecipitation was not preferential to the

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

absence of Man-6-P-containing ligands [25]. York
et al. [24] demonstrated, by sucrose gradient sedimen-
tation and gel filtration, that the receptor bound to a
multivalent Man-6-P-containing ligand, b-glucuroni-
dase, exhibited a sedimentation coefficient and Stokes
radius that were consistent with a complex of two
receptor molecules plus one molecule of ligand. How-
ever, when these experiments were performed with
IGF-II, the receptor appeared to exist as a monomer.
Internalization experiments performed with [
125
I]IGF-
II in the presence of b-glucuronidase revealed that
b-glucuronidase accelerated the rate of IGF-II uptake,
suggesting that intermolecular cross-linking of recep-
tors enhanced receptor endocytosis [24]. Thus, all the
available data are consistent with the conclusion that
the M6P⁄ IGF2R functions as a dimer in high-affinity
binding of Man-6-P-bearing ligands, but possibly not
for IGF-II. The present study, employing soluble
forms of the receptor [25,26,34], indicates that the
ectodomain of the receptor is capable of dimer forma-
tion in the absence of ligand.
Affinity cross-linking of [
125
I]IGF-II to a mutant
repeat 11 mini-receptor revealed that the I1572T muta-
tion completely abolished IGF-II binding [31]. This
finding was confirmed by Linnell et al. [22] by utilizing
surface plasmon resonance of a truncated receptor

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

notion that this structure is the receptor’s normal func-
tional state.
The most interesting and unexpected finding of the
present study is that asymmetric heterodimers (i.e. hav-
ing different epitope tags, of which only one is tethered
to the resin bead) demonstrate complex binding
behavior. Dimers of this type exhibit the predicted
amount of binding only if the heterodimer is tethered
by the wild-type partner. By contrast, we found that, if
the heterodimer is tethered to the resin by the mutant
partner, the amount of binding observed is substan-
tially less than expected. This complex binding behav-
ior observed with IGF-II binding must only be a local
effect because binding of PMP-BSA, which binds to
other sites in the ectodomain of these heterodimers,
resulted in the predicted amount of ligand binding.
This loss of binding function observed with asym-
metric heterodimers may be due to deformation of the
dimeric structure. One possible explanation for failure
to form a dimer of correct structure could be steric
hindrance between the Myc tag and the M2 resin bead.
This is envisioned to cause the Myc-tagged receptor
partner to be bent outward away from the tethered
FLAG-tagged partner, potentially resulting in distor-
tion of the IGF-II binding pocket and a consequent
reduced ability to bind IGF-II. This effect is likely not
due to reduced contact between repeats 11 and 13
because repeat 11 is capable of binding IGF-II even in
the absence of repeat 13 [20,22]. These data suggest
that the failure to tether the tail of the extracyto-

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

leads to the question as to whether the RfiA mutation
actually causes a loss of binding energy sufficient to
abrogate Man-6-P binding or whether the mutated
binding pocket becomes distorted, preventing Man-6-P
binding. Glucose 6-phosphate (Glc-6-P) is unable to
bind to the two Man-6-P binding sites of the receptor
[17]. The only difference between Glc-6-P and Man-6-P
is the position of the 2-hydroxyl group. The equatorial
2-hydroxyl group of Glc-6-P is not in the correct posi-
tion to make the necessary hydrogen bonds with the crit-
ical Arg; however, the axial 2-hydroxyl of Man-6-P is.
Therefore it appears that the binding energy of the site
must be decreased with the RfiA mutation, preventing
Man-6-P from binding to the Man-6-P binding sites.
Based on this analysis and the need for cooperative
interaction between two binding sites, we expected the
wild-type ⁄ R2A heterodimers to show equivalent B
max
with reduced affinity; however, ligand binding analysis
(data not shown) revealed a 50% decrease in B
max
,
whereas the affinity remained unchanged. This surpris-
ing finding led us to hypothesize that the large multi-
valent Man-6-P-based ligands such as PMP-BSA can
bind bivalently to wild-type subunits of heterodimeric
receptors juxtaposed on the resin. Whether these repre-
sent nearby heterodimers or receptors interacting in
large oligomeric clusters is not yet clear. It is known
from previous studies that the M6P ⁄ IGF2R is concen-

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-

able in a mixture of mutant ⁄ wild-type receptors.
Tethering to the resin of asymmetric heterodimers sug-
gests the importance of anchoring the tail of the ecto-
domain for both IGF-II and Man-6-P ligand binding.
Receptor binding on resin resembles a patchwork
model, where a multivalent ligand can cross-bridge
between nearby wild-type sites to achieve high-affinity
binding. Additionally, further studies are needed to
obtain more information on the structure of the
M6P ⁄ IGF2R ectodomain, particularly toward the
C-terminal repeat 15 and its interaction with the mem-
brane, to assess whether there are important associated
proteins or receptor-lipid interactions that cannot be
mimicked using epitope-tagged portions of the ectodo-
main. Finally, experiments in whole cells should
address the possibility that the receptor can associate
in patches during endocytosis.
Experimental procedures
Materials
d-Man-6-P disodium salt, anti-FLAG M2 Ig anti-FLAG
M2-agarose affinity gel and the protein G-Sepharose
reagents were obtained from Sigma (St Louis, MO, USA).
Anti-Myc 9E10 Ig was purchased from Upstate Biotech-
nology, Inc. (Hercules, CA, USA) or the University of
Nebraska Medical Center Monoclonal Antibody Facility
(Omaha, NE, USA). Anti-HA Ig was purchased from
Roche Diagnostics Corporation (Indianapolis, IN, USA).
Rabbit anti-(mouse IgG) was obtained from Dako Corp.
(Carpinteria, CA, USA) and carrier-free Na[
125

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

repeats of the M6P ⁄ IGF2R followed by a FLAG epitope
tag (DYKDDDDK), called 1-15F, was engineered from the
hIGF2R cDNA as described previously [40]. Two similar
receptors, 1-15Myc and 1-15HA, were also generated using
the same strategy with a COOH-terminal Myc (ME-
QKLISEEDLN) or HA (YPYDVPDYA) epitope tag engi-
neered in place of the FLAG tag [25]. Site-directed
mutagenesis was carried out using the QuikChangeÔ muta-
genesis kit from Stratagene (La Jolla, CA, USA). Pfu-direc-
ted thermal cycling was conducted with a fragment of the
human M6P ⁄ IGF2R cDNA encompassing two PflMI sites
(nucleotides 3847–6315) in pCRII (Invitrogen, Carlsbad,
CA, USA). Complementary primer pairs corresponding to
nucleotides 4840–4874 substituting T to C at nucleotide
4862 creating the Ile1572>Thr (I1572T) mutation. The
resultant product was digested with PflMI and subcloned
into a shuttle vector containing the M6P ⁄ IGF2R cDNA.
The presence of the mutation was confirmed by sequence
analysis. Finally, the mutant construct was digested with
EagI, and the 5.2 kb fragment was subcloned into the
pCMV5 vector containing the FLAG-tagged (1-15F), Myc-
tagged (1-15Myc) or HA-tagged (1-15HA) human receptor
constructs creating the 1-15F I ⁄ T, 1-15Myc I ⁄ T and
1-15HA I ⁄ T mutant receptors, respectively.
Soluble, FLAG- or Myc-tagged mini-receptors that were
mutant for residues in the repeats 3 and 9 Man-6-P binding
domains (R426A and R1325A, respectively) were prepared
as follows: nucleotides 100–3242 of the M6P ⁄ IGF2R
cDNA served as a template for amplification using a 5¢-pri-
mer containing an EcoRI restriction site preceding the

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

mutations at both repeats 3 and 9 (1-15F R2A and
1-15Myc R2A).
Expression and analysis of epitope-tagged
soluble receptors
Transient expression of soluble receptors was performed in
293T human embryonic kidney cells cultured in DMEM
supplemented with 5% fetal bovine serum and 5 lgÆlL
)1
gentamycin at 37 °C in a humidified atmosphere of 5%
CO
2
⁄ 95% air. Transfection was carried out by a modifica-
tion of the calcium phosphate method previously described
[42]. Cells were harvested 3 days after transfection and
lysates were prepared by solubilization with 50 mm Hepes,
pH 7.4, 1% Triton X-100, and 1 mm MgCl
2
, as described
previously [31]. After lysates were prepared, 25 lL aliquots
were electrophoresed on a 6% SDS ⁄ PAGE gel under reduc-
ing conditions and transferred to BA85 nitrocellulose paper
(Schleicher & Schuell Bioscience, Dassel, Germany). The
blots were blocked with 4% nonfat milk in 50 mm Hepes,
pH 7.4, 150 mm NaCl, 0.1% Tween 20 and probed with
either anti-FLAG M2 Ig (1 : 2000), anti-Myc 9E10 Ig
(1 : 500) or anti-HA Ig (1 : 250) followed by a secondary
rabbit anti-(mouse IgG). The resulting antibody complex
was developed with [
125
I]protein A and detected by means

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

end-over-end mixer. The resin was collected by centrifuga-
tion at 13 000 g for 20 s. The resulting resin pellets were
washed three times with 1 mL of HBS containing 0.05%
Triton X-100 (HBST). The resin pellets were heated to
100 °C for 5 min and electrophoresed on a 6% SDS ⁄ PAGE
gel under reducing conditions and transferred to BA85
nitrocellulose paper. The blots were probed with either
anti-FLAG M2, anti-Myc 9E10 or anti-HA Igs developed
with [
125
I]protein A, followed by autoradiography and
PhosphorImager analysis.
Co-immunoprecipitation of soluble receptors
with anti-Myc 9E10 Ig coupled to protein
G-Sepharose
Aliquots of 293T cell lysates, containing equimolar
amounts of expressed Myc-tagged soluble receptors, were
incubated with 1 lL (1 : 100) of anti-Myc 9E10 Ig in HBST
for 16 h at 4 °C. Protein G-Sepharose, preblocked by wash-
ing in HBS + 1% BSA, in 6 lL of packed resin was intro-
duced into the lysate-antibody mixture and allowed to
incubate for 3 h at 4 °C on an end-over-end mixer. The
resin was collected by centrifugation at 13 000 g for 20 s.
The resulting resin pellets were washed three times with
HBST. The pellets were heated to 100 °C for 5 min in
sample buffer and electrophoresed on a 6% SDS ⁄ PAGE
gel under reducing conditions and transferred to BA85
nitrocellulose paper. The blots were probed with either
anti-FLAG M2 or anti-Myc 9E10 Igs developed with
[

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-

ing was determined by subtracting c.p.m. ligand bound
in replicate reactions carried out in the presence of 1 lm
IGF-II. Binding of [
125
I]PMP-BSA was measured under
similar conditions using 1 nm [
125
I]PMP-BSA in the pres-
ence or absence of 5 mm Man-6-P to compensate for non-
specific binding. The data were graphed and analyzed using
graphpad prismÔ (Graphpad Software, San Diego, CA,
USA). Ligand binding parameters were measured by com-
petitive binding analysis. Equal amounts of the receptor
constructs were immunoadsorbed to M2 resin and incu-
bated with 1 nm [
125
I]PMP-BSA in the presence of increas-
ing concentrations of unlabeled PMP-BSA (0–500 nm)at
4 °C for 3 h. The resin pellets were then washed and
counted as described above. The data were fit to a model
for one-site and two-site competitive binding using graph-
pad prismÔ.
Acknowledgements
We thank Dr William S. Sly for providing the human
M6P ⁄ IGF2R cDNA, Dr David W. Russell for pro-
viding pCMV5 and The Rockefeller University for
permission to use the 293T cells. We are grateful to
Margaret H. Niedenthal of Lilly Research Laborato-
ries for providing the IGF-II. We thank Barbara
Switzer and the University of Nebraska Medical Cen-

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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