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 bindin
Trang 1heteromeric complexes of the mannose
6-phosphate/insulin-like growth factor II receptor
Michelle A Hartman1, Jodi L Kreiling2, James C Byrd1and Richard G MacDonald1
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: rgmacdon@unmc.edu
(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.
Trang 2milieu, 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
Trang 3Transient 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-eight-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 [125I]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 [125I]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
H 2 N
H 2 N
H 2 N
*
*
Construct name:
1-15F
1-15F I/T
1-15Myc
1-15Myc I/T
3
5 1- 1 F 1-15F
I/T -15 1 My c 1-15 M c T y /
IGF-II
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 [125I]PMP-BSA (B) or [125I]IGF-II (C) and developed
by autoradiography The autoradiograms of representative blots are
shown.
Trang 4able 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
I-IGF-II binding c.p.m × 10
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 ‘125I-IGF-II binding’.]
Trang 5receptors (Fig 3E) Binding of [125I]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 formamuta-tion of oligomers
that are functional in phosphomannosyl ligand binding
and establishing a baseline of ligand binding function
(Fig 3E)
Binding of [125I]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: αα -FLAG
IB: α -FLAG
IB: α -Myc
IB: IP: α -Myc 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
0
10
20
30
40
I-IGF-II binding c.p.m × 10
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
5 m M Man-6-P or 1 l M IGF-II was subtracted from each binding reaction to determine the specific binding for [125I]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 ‘125I-PMP-BSA binding’ was corrected to
‘ 125 I-IGF-II binding’.]
Trang 6receptors 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 [125I]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 [125I]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
I-IGF-II binding c.p.m × 10
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’.]
Trang 7anchored 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 [125I]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 [125I]PMP-BSA
to immunoprecipitated mini-receptors was measured to assess whether the presence of the mutant 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
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
I-IGF-II binding c.p.m × 10
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.
Trang 8be 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 [125I]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
1-15Myc
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: IP: α -Myc α -FLAG
I-IGF-II binding c.p.m × 10
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 n M [ 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
‘125I-IGF-II binding’.]
Trang 9individually immunoprecipitated, FLAG-tagged
mini-receptors
Binding of [125I]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 sbind-ingle 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
Trang 10demon-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 [125I]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