An Insulin-Like Growth Factor II (IGF-II) Affinity-Enhancing Domain Localized within Extracytoplasmic Repeat 13 of the IGF-II/Mannose 6-Phosphate Receptor
Gayathri R. Devi,
James C. Byrd,
Dorothy H. Slentz and
Richard G. MacDonald
Department of Biochemistry and Molecular Biology University of
Nebraska Medical Center Omaha, Nebraska 68198-4525
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ABSTRACT
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Insulin-like growth factor II (IGF-II) and
phosphomannosylated glycoproteins bind to distinct sites on the same
receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of
truncated receptors (minireceptors) has been used to map the IGF-II
binding site within the receptors extracytoplasmic domain, which
consists of 15 homologous repeats. A minireceptor consisting of repeat
11 contained the minimal elements for binding IGF-II, but with 5- to
10-fold lower relative binding affinity than the full-length receptor.
We hypothesized that the complete, high-affinity IGF-II binding site is
formed by interaction between the primary site in repeat 11 and a
putative affinity-enhancing domain. To determine the minimum portion of
the IGF2Rs extracytoplasmic domain needed for expression of
high-affinity IGF-II binding, a nested set of FLAG epitope-tagged
minireceptors encompassing repeats 11 through 15 was prepared and
transiently expressed in 293T cells. Minireceptors containing repeats
1113 or 1115 exhibited high affinity, comparable to the full-length
receptor (IC50 = 12
nM), whereas constructs containing repeat 11
only or repeats 1112 did not (IC50 = 1020
nM). These data suggested that the
affinity-enhancing domain is located within repeat 13, which contains a
unique 43-residue insert that has
50% sequence identity to the type
II repeat of fibronectin. Although a repeat 13 minireceptor did not
bind IGF-II on its own, an 1113 minireceptor containing a deletion of
the 43-residue insert exhibited low IGF-II binding affinity
(IC50 = 1020 nM).
Expression of mutant receptors from a full-length IGF2R construct
bearing a deletion of the 43-residue insert was very low relative to
wild type. Depletion assays using IGF-II-Sepharose showed that the
mutant receptor had lower affinity for IGF-II than the wild-type
receptor. This study reveals that two independent receptor domains are
involved in the formation of a high-affinity binding site for IGF-II,
and that a complete repeat 13 is required for high-affinity IGF-II
binding.
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INTRODUCTION
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The insulin-like growth factor-II (IGF-II)/mannose 6-phosphate
(Man-6-P) receptor (IGF2R) is a 300-kDa transmembrane protein that
contains binding sites for both IGF-II and phosphomannosylated
glycoproteins (1, 2). The IGF2R is composed of a relatively short
C-terminal cytoplasmic domain connected via a single transmembrane
domain to the extracytoplasmic region consisting of 15 homologous
repeats (3, 4, 5). These repeats exhibit
20% identity to each other
and to the extracytoplasmic region of the cation-dependent Man-6-P
receptor (5). An insertion of a 43-residue segment after the fourth
cysteine residue in repeat 13 is the only major interruption of the
repetitive structure (6). This insert exhibits sequence identity to the
type II domain of fibronectin, a disulfide-linked structure that is
repeated in tandem fashion in its collagen-binding domain (7). A type
II domain also occurs once in factor XII (8) and as a tandem repeat in
the bovine seminal fluid proteins PDC-109 (9) and BSP-A3
(10), as well as in 72,000 and 92,000 Mr forms of type IV
collagenase (11).
The Man-6-P binding sites that have been localized to repeats 13 and
79 of the IGF2R bind with a stoichiometry of 2 mol of Man-6-P/mol of
receptor (12). The human, bovine, rat, and opossum (13) receptors bind
both IGF-II and Man-6-P, whereas the chicken and frog homologs do not
bind IGF-II (1, 2, 14, 15). The IGF2R transports lysosomal enzymes and
other Man-6-P-bearing glycoproteins from the locus of posttranslational
processing in the Golgi to an acidic prelysosomal compartment (1, 16, 17). In the rat adipocyte model, molecules of IGF-II that bind to the
IGF2R at the cell surface are rapidly internalized and transported to
lysosomes for degradation (18). Current evidence suggests that the
IGF2R does not mediate a signal transduction event in response to
IGF-II binding, and that most of the anabolic, mitogenic, and
antiapoptotic activities of IGF-II are mediated by binding to the IGF-I
receptor [IGF1R (19, 20)].
Gene knock-out experiments and analysis of IGF2R expression in
human cancer have provided new insights into the IGF-II binding
function of the receptor. The M6p/Igf2r locus is paternally
imprinted in mice, and embryos inheriting a maternally derived deletion
of the Tme region encompassing M6p/Igf2r die at
day 15 of gestation (21). Subsequently, Lau et al. (22)
showed that mice inheriting a disrupted M6p/Igf2r maternal
allelle died near birth. Lethality associated with the
M6p/Igf2r- phenotype is related to the
Igf2 gene product, as the phenotype could be rescued by
knock-out of either Igf2 or Igf1R on the
M6p/Igf2r nullizygous background (23, 24). These findings
suggest that IGF-II binding by the IGF2R followed by internalization
and degradation of the ligand is a key regulatory mechanism for
modulation of IGF-II levels during development. If IGF-II is mitogenic
and even tumorigenic when expressed at high levels in tumor cells (25),
then it follows that the IGF2R could serve as a suppressor of
IGF-II-dependent tumors (26, 27, 28). Although M6P/IGF2R is not
imprinted in all humans (29), loss of heterozygosity and mutations in
the remaining allele at the M6P/IGF2R locus have been
observed in hepatocellular carcinomas (30, 31) and in breast tumors
(32). In addition, M6P/IGF2R mutations have been observed in
colorectal, gastric, and endometrial tumors exhibiting microsatellite
instability (26, 27). Collectively, these studies support the
hypothesis that the IGF2R functions as a tumor suppressor in those
tissues. Regulation of IGF-II levels is critical for normal growth
control, whereas subversion of this control mechanism by reduction in
IGF2R levels or possibly via alterations in the receptors IGF-II
binding properties may be key in tumorigenesis.
The potential role of the IGF2R in human cancer has enhanced interest
in understanding the receptors ligand-binding properties. Recent
studies (33, 34, 35, 36) using truncated forms of the extracytoplasmic region
of the human IGF2R have shown that repeat 11 contains the elements
necessary for formation of a minimal IGF-II binding site, and that
ligand binding specificity resides within the NH2-terminal
half of repeat 11. However, it has also become clear that the truncated
receptors encompassing repeats 811 have 5- to 10-fold lower IGF-II
binding affinity than the full-length receptor (35). On the other hand,
in a study by Schmidt et al. (36), both the full-length
receptor and a truncated receptor encompasssing repeats 1015
exhibited equivalent IC50 values for displacement of
radiolabeled IGF-II binding. Those studies imply the existence of a
second region of the extracytoplasmic domain, possibly located
between repeats 1215 of the IGF2R, that may cooperate with the
principal site to enhance the affinity of IGF-II binding.
To localize the minimum portion of the IGF2Rs extracytoplasmic domain
needed for expression of high-affinity IGF-II binding, a series of cDNA
constructs encoding a nested set of secreted, truncated forms of the
human IGF2R was prepared. These minireceptors were transiently
expressed in 293T cells and analyzed for their abilities to bind
IGF-II. These studies localize the affinity-enhancing domain to repeat
13 of the IGF2Rs extracellular region and implicate the 43-residue
type II domain within that region as required for conferring high
affinity on the IGF-II binding site.
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RESULTS
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Expression of IGF2R Minireceptor Constructs
A schematic representation of the strategy used to prepare the
IGF2R cDNA constructs, based on a published approach (35), is shown in
Fig. 1A
. An EcoRI site located
at nucleotide (nt) 475 of the human IGF2R cDNA that bisects the region
encoding the first extracytoplasmic repeat was used for expression of
the natural signal sequence and the NH2-terminal half
(first 71 residues) of repeat 1 in all the minireceptor constructs. The
indicated repeats between 11 and 15 were amplified by PCR and inserted
into a modified pCMV5 vector bearing the 5'-receptor cDNA fragment. All
the minireceptor constructs were fused to an eight-residue,
COOH-terminal FLAG epitope tag. These minireceptor constructs were
transiently expressed in 293T cells, and the cell lysates were
immunoblotted with the anti-FLAG M2 monoclonal antibody to confirm
synthesis of the minireceptors (Fig. 1B
). Small discrepancies between
the estimated Mr for the 1112 and 1113 minireceptors
relative to the predicted Mr may have arisen from N-linked
glycosylation. There is one potential N-linked glycosylation site in
repeat 11, whereas there are two such sites each in repeat 12 and
repeat 13 (3, 4).

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Figure 1. Design and Expression of IGF2R Minireceptors
A, The cDNA cassettes for preparation of truncated receptor constructs
were synthesized by a series of PCR reactions using the human IGF2R
cDNA as template and incorporating sequence encoding a COOH-terminal
FLAG epitope tag. Each cassette begins at the N-terminal residue of
repeat 11 and ends at the C-terminal residue of repeats 11 through 15
(open squares). These cassettes were subcloned
downstream of sequence encoding the signal peptide (darkly
stippled square) and the N-terminal 70 residues of repeat 1 of
the human IGF2R cDNA (lightly stippled square) in pCMV5.
Molecular weights of the resultant minireceptors have been predicted
according to their amino acid contents, excluding the potential
contributions of N-linked oligosaccharide chains. B, Lysates prepared
from 293T cells on the sixth day after transfection with the various
minireceptor constructs were assayed for expression of the IGF2R
minireceptors by immunoblotting using the M2 monoclonal antibody
coupled to ECL. Molecular weights of the immunoreactive species
indicated along the right-hand border were calculated
from the electrophoretic mobilities relative to standard proteins, as
indicated at the left.
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Analysis of IGF-II Binding to IGF2R Minireceptors
To determine whether the regions encompassed by the minireceptors
contained a functional IGF-II binding/cross-linking site, the purified
minireceptors immunoadsorbed to M2 affinity resin were incubated with 2
nM [125I]IGF-II plus 100 nM
unlabeled IGF-I. Unlabeled IGF-I was included in the reaction to
competitively displace radiolabeled IGF-II from, and thereby eliminate
interference by, IGF binding proteins (IGFBPs). The resin-bound
IGF-II-minireceptor complexes were then affinity cross-linked with
disuccinimidyl suberate and analyzed by SDS-PAGE. The data in Fig. 2
demonstrate that
[125I]IGF-II is specifically cross-linked to all the
minireceptors in the cell lysates. That binding to these minireceptors
is specific is supported by the disappearance of the major radioactive
bands on incubation with excess unlabeled IGF-II during affinity
labeling.

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Figure 2. Affinity Labeling of IGF2R Minireceptors with
[125I]IGF-II
Minireceptors were immunoadsorbed from lysates prepared on day 6
posttransfection of 293T cells, using M2 antibody resin. The purified
minireceptor resins were incubated with 2 nM radiolabeled
IGF-II with or without 1 µM unlabeled IGF-II for 3 h
at 3 C and then cross-linked with 0.25 mM DSS for 30 min at
22 C. The affinity-labeled minireceptors were separated on a 10% SDS
polyacrylamide gel. An autoradiogram of the dried gel is shown. Values
associated with the arrows along the right-hand border
of the gel indicate calculated molecular weights of the major
radioactive species.
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For estimation of the affinity of IGF-II binding, resin-bound
minireceptors were affinity cross-linked with 2 nM
[125I]IGF-II plus 100 nM unlabeled IGF-I in
the presence of increasing amounts of unlabeled IGF-II from 1
nM to 500 nM (Fig. 3
). A gradual displacement of
[125I]IGF-II labeling of the minireceptor constructs 11,
1112, 1113, and 1115 was observed with increasing concentrations
of unlabeled IGF-II (Fig. 3
). However, complete displacement of the
major radioactive band in the case of the 1113 and 1115
minireceptors occurred at lower concentrations of unlabeled IGF-II
(12 nM), when compared with the minireceptors
encompassing repeat 11 (1020 nM) or repeats 1112
(1530 nM). The top panel of Fig. 3
shows
the results obtained when this same analysis was done with the
full-length IGF2R present in plasma membranes from rat BRL-3A hepatoma
cells.

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Figure 3. Displacement Analysis of [125I]IGF-II
Cross-linked to IGF2R Minireceptors
IGF2R minireceptors were immunoadsorbed from cell lysate preparations
of transfected 293T cells, using M2 antibody resin. BRL 3A plasma
membranes for analysis of the full-length receptor, designated as
Receptor, were prepared as described in Materials
and Methods. All samples were incubated with 2 nM
radiolabeled IGF-II in the presence of the indicated concentrations of
unlabeled IGF-II. Cross-linking was then done with DSS, followed by
SDS-PAGE. Autoradiograms of the dried gels are shown.
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Quantitative analysis of the radiolabeled bands in these gels was done
by PhosphorImager, and the data were plotted as a function of IGF-II
concentration to construct [125I]IGF-II displacement
curves (Fig. 4
). The curves for
minireceptors 11 and 1112 were shifted to the right relative to those
of the full-length receptor and minireceptors 1113 and 1115.
Calculation of the unlabeled IGF-II concentrations required to achieve
50% displacement of binding (IC50) indicated that
minireceptors 11 and 1112 had IC50 values of 1520
nM, which were approximately 1 order of magnitude higher
than those of minireceptors 1113, 1115, and the full-length
receptor [IC50 values in the range 15 nM
(Table 1
)].

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Figure 4. Quantitative Analysis of Radiolabeled
125I-IGF-II Displacement from IGF2R Minireceptors
The gels shown in Fig. 3 were analyzed by PhosphorImager for
quantitative estimation of the radioactivity associated with the
individual bands. The data for each gel have been expressed as a
percentage of the maximal band intensity.
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Deletion of the Type II Repeat in Repeat 13 Reduces Affinity of the
1113 Minireceptor
Since the 1113 and 1115 minireceptors showed high affinity
comparable to the full-length IGF2R and the 1112 minireceptor did
not, we hypothesized that the unique 43-residue insert in repeat 13
might be involved in the formation of the affinity-enhancing domain. To
test this hypothesis, two minireceptor constructs were prepared (Fig. 5A
), using a strategy similar to that
described in the legend to Fig. 1
: minireceptor construct
1113(
43), which contained repeats 1113 but lacked the 43-residue
insert, as well as a minireceptor encompassing only repeat 13. These
constructs were transiently expressed in 293T cells, and cell lysates
were subjected to SDS-PAGE followed by immunoblotting with the
anti-FLAG M2 monoclonal antibody (Fig. 5B
). A small discrepancy between
the estimated Mr for the 1113(
43) and repeat 13
minireceptors relative to the predicted Mr may have arisen
from N-linked glycosylation at two potential sites in repeat 13 (3, 4).
To determine whether the regions encompassed by these two minireceptors
contained a functional IGF-II binding/cross-linking site,
[125I]IGF-II affinity labeling of the purified
minireceptors was carried out. The data in Fig. 6
demonstrate that
[125I]IGF-II did not cross-link to the repeat 13
minireceptor. When compared with an equivalent amount of the wild-type
(WT) 1113 minireceptor, the 1113(
43) minireceptor showed a
specific, but very low-intensity cross-linked band. Estimation of
IGF-II binding affinity revealed the IC50 value estimated
for the 1113(
43)-IGF-II complex occurred at higher concentrations
of unlabeled IGF-II (1020 nM) relative to that of the
1113-IGF-II complex (IC50 = 1.54 nM, Figs. 3
and 4
). Experiments measuring direct binding of 2 nM
[125I]IGF-II to these minireceptors revealed no
detectable binding to the repeat 13 minireceptor and very poor binding
to the 1113(
43) minireceptor relative to the WT 1113
minireceptor (data not shown).

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Figure 6. Affinity Labeling of Minireceptors 11, 13, and
1113( 43) with [125I]IGF-II
Minireceptors 1113( 43) and 13 were immunoadsorbed from lysates
prepared on day 6 after transfection of 293T cells, using M2 antibody
resin. The purified minireceptor resins were incubated with 2
nM radiolabeled IGF-II with or without 1 µM
unlabeled IGF-II for 3 h at 3 C and then cross-linked with DSS.
The affinity-labeled minireceptors were subjected to SDS-PAGE; an
autoradiogram of the dried gel is shown.
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Characterization of a Full-Length IGF2R Construct Bearing a
Deletion of the Type II Repeat
A full-length IGF2R cDNA mutated to produce a deletion of the
43-residue insert (
43)IGF2R was transfected into 293T cells for
analysis of its IGF-II binding characteristics. However, there was no
detectable overexpression of the full-length (
43)IGF2R above the
background of the band attributable to the endogenous 293T cell
receptor (data not shown). A parallel transfection with a WT,
full-length IGF2R construct produced 3-fold overexpression of the
Mr 300,000 IGF2R species. Similar results were
obtained upon several repeat transfections in 293T cells and in other
cell lines, such as COS-7 and LRec- mouse L cells.
Synthesis of the (
43)IGF2R mRNA was confirmed by RT-PCR of total RNA
from untransfected 293T cells vs. cells transfected with the
full-length WT IGF2R cDNA, suggesting that the (
43)IGF2R construct
allows transcription of a stable mRNA, but that the mutant protein
accumulates to very low levels in the cells (data not shown).
The (
43)IGF2R encoded by the original construct retained a Gly-Thr
dipeptide remnant of the 43-residue type II domain. To rule out the
possibility that this incomplete deletion destabilized the receptor, a
second full-length construct bearing a complete deletion of the type II
domain was prepared by an entirely different mutagenesis strategy.
Expression of the mutant receptor from this new construct in 293T cells
was also extremely low relative to its WT counterpart. Nevertheless, we
were able to detect the presence of the deletion mutant because of a
c-myc epitope tag that was added to the COOH-terminal ends
of these new constructs (Fig. 7A
). The
band corresponding to (
43)IGF2R-myc was of slightly lower apparent
Mr than that of the WT IGF2R-myc, as expected from the
deletion of the 43-residue type II domain. Quantitative analysis of
band intensities from Western blots confirmed that expression of the
mutant receptor was very low, averaging about 4% of WT in several
different experiments.
We had intended to exploit the presence of the c-myc epitope
tag to selectively immunoprecipitate exogenously expressed receptors so
that their ligand-binding characteristics could be analyzed in the
absence of endogenous receptor background. Unfortunately, the receptors
tagged at the COOH-terminal end could not be immunoprecipitated from
detergent solutions with anti-myc antibody under a variety
of conditions (data not shown).
Therefore, to assess the IGF-II binding properties of the
43 and WT
IGF2R-myc receptors, an IGF-II-Sepharose-based affinity-depletion assay
was used. Receptors solubilized from 293T plasma membranes were
incubated either with the IGF-II-Sepharose affinity resin or blank
Sepharose, and then assayed for the amount of unbound receptor,
i.e. that remaining in the postresin supernatant, by
SDS-PAGE followed by immunoblotting with anti-myc antibody
(Fig. 7B
). In three replicate experiments, a mean of 28% of the WT
IGF2R-myc remained in solution after incubation with the
IGF-II-Sepharose resin, whereas 108% remained after exposure to the
blank Sepharose. The difference between these values is taken as an
indicator of the affinity of the myc-tagged receptors for
the immobilized IGF-II. In contrast, there was substantially less
difference between the amounts of (
43)IGF2R-myc remaining in
solution after incubation with IGF-II-Sepharose (83%) vs.
blank resin (108%). These data indicate reduced IGF-II affinity of
(
43)IGF2R-myc relative to WT IGF2R-myc under these conditions,
suggesting a substantial impairment of the IGF-II binding function of
the receptor bearing the deletion of the 43-residue type II domain in
repeat 13.
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DISCUSSION
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We report herein that two nonadjacent domains, the primary binding
site and an affinity-enhancing domain, interact to form the
high-affinity IGF-II binding site of the human IGF2R. Our principal
conclusion is that the affinity-enhancing domain is located in repeat
13 of the extracytoplasmic region of the receptor. The observation that
the 1113(
43) minireceptor bearing a deletion of the unique
43-residue insert exhibited low IGF-II binding affinity relative to the
WT 1113 minireceptor strongly suggests that the amino acid residues
responsible for the formation of an affinity-enhancing domain are
encompassed within repeat 13. The affinity-enhancing domain in repeat
13 is clearly not part of the primary IGF-II binding site located in
repeat 11, as previous work (33, 34, 35) has shown that repeat 11 contains
the minimal elements necessary for IGF-II binding. However, ours is the
first study revealing the involvement of the unique 43-residue insert
in enhancing the IGF-II binding affinity of the human IGF2R.
Our conclusions are consistent with the predictions of the study by
Schmidt et al. (36) and our previous work (35) involving
mapping of the primary IGF-II binding site: that the IGF-II binding
domain of the receptor is bipartite, in a manner reminiscent of the
bivalent Man-6-P binding domain of the IGF2R. Each of the two Man-6-P
binding sites located in different regions of the receptor, in repeats
3 and 9 (13, 37), are capable of binding a single phosphomannosyl
moiety with low affinity (12). Simultaneous interaction of both Man-6-P
binding sites with oligosaccharide(s) having two phosphomannosyl
moieties provides for highest affinity binding (12). With respect to
IGF-II binding to the IGF2R, the "half-sites" are clearly not
equivalent. We postulate that two mechanisms that are not mutually
exclusive may underlie the interplay between the two independent
domains in the ligand-binding process: 1) that both repeats 11 and 13
possess ligand-binding determinants, and/or 2) that conformational
effects imposed on repeat 11 by repeat 13 are required for strong
ligand binding to repeat 11. Our data suggest that repeat 13
does not contain a secondary IGF-II binding site, because a repeat 13
minireceptor construct could not cross-link or bind to IGF-II. However,
this result must be interpreted with caution because the noncovalent
interactions capable of enhancing affinity of the primary binding site
by only 1 order of magnitude may not allow for formation of a
detectable complex between IGF-II and the repeat 13 region alone.
Another possible interpretation of our data is that the
affinity-enhancing domain stabilizes IGF-II interaction with the
primary binding site by making noncovalent contacts with repeat 11 or
with both repeat 11 and the bound ligand. In the latter instance, it
must be envisioned that interactions between repeat 13 and IGF-II are
few or weak.
It has been shown that the bovine seminal plasma proteins PDC-109 and
BSP-A3, each of which contains the 43-residue insert as a
tandem repeat, are capable of binding IGF-II (38). However, this
binding is of low affinity (IC50 =
25 nM),
and it is unknown whether their type II repeat structures are actually
involved in IGF-II binding. Constantine et al. (39)
characterized the solution conformation of the type II domain of
PDC-109 by 1H-NMR, and they concluded that several aromatic
and acidic residues were implicated in binding to collagen. It is of
interest to note that those five residues are extremely well conserved
within the primary structures of the mammalian IGF2R proteins sequenced
to date from various species (3, 4, 5, 40). Equally well conserved between
all such proteins are the four Cys residues that presumably form
disulfide bonds within the type II domain. These properties are shared
by the the chicken CI-MPR, which does not bind IGF-II because of a
major divergence in sequence in the repeat 11 region between the
chicken receptor and its mammalian counterparts (41). The rat IGF2R
does not bind to collagen or gelatin (R.G. MacDonald, unpublished
results), suggesting the possibility that substitution of residues
within the type II domain may have produced different binding
specificities for this domain in the various proteins in which it
occurs. However, the data available (Ref. 38 , and this study) do not
support the notion that the type II domain is involved in extensive
binding interactions with IGF-II. In the present studies, a full-length
IGF2R construct bearing a deletion of the 43-residue type II repeat was
expressed at very low levels in 293T cells, and that protein had no
detectable affinity for IGF-II in a depletion assay. Although a
minireceptor construct bearing the same mutation produced robust
expression of an apparently stable protein, the 1113(
43)
minireceptor had very low IGF-II binding affinity relative to the WT
1113 minireceptor. Repeat 13 of the IGF2R, particularly the
43-residue insert, may be a critical conformational component of the
receptor, through noncovalent interaction with neighboring domains such
as repeat 11 and possibly repeat 12. Our findings indicate that one
manifestation of its putative structural role is an enhancement of
IGF-II binding affinity. We may speculate that deletion of the type II
repeat causes local polypeptide misfolding, altered receptor
conformation, or enhanced susceptibility to proteolysis, but those
questions remain for future experiments. An alternative strategy for
testing the role of the type II repeat in IGF-II binding and
conformational stabilization of the IGF2R would be domain swapping of
the type II repeats between PDC-109 or fibronectin and that of the
receptors 13th repeat. Currently, we are involved in such experiments
as well as detailed analysis of the mechanism of interaction of the
three adjacent repeats, 11, 12 and 13, in the formation of the
high-affinity IGF-II binding site.
The participation of two domains within the same protein in ligand
interaction is not unusual. The crystal structure of the trimeric
GH-receptor complex revealed that the hormone-receptor interface
includes similar residues from two distinct receptor extracellular
domains as well as from the intervening COOH-terminal linker regions
(42). For a number of other proteins such as the urokinase receptor
(43), talin (44), the Na,K-ATPase
-subunit (45), coagulation factor
VIIa (46), studies with isolated domains and proteolytic fragments have
suggested the involvement of two or more domains in formation of ligand
contact regions.
In summary, the present work provides strong evidence for localization
of an IGF-II affinity-enhancing domain to repeat 13 of the
extracellular region of the IGF2R. The function of this domain is of
marked physiological significance, because it is responsible for a
10-fold enhancement of IGF-II binding affinity. In the context of the
IGF2Rs proposed role in regulating tissue IGF-II levels during normal
development and its potential tumor suppressor activity, a decrease in
IGF-II binding affinity would translate into an increase in local
IGF-II concentration. The precise magnitude of this increase and the
increment in IGF-II availability for binding to the IGF1R would be
difficult to calculate due to confounding factors such as diffusion and
binding of some of the excess IGF-II by IGFBPs. Identification of this
affinity-enhancing domain makes it possible to search for mutations
within that region in cancers having loss of heterozygosity at the
M6P/IGF2R locus, which would improve understanding the
mechanism of IGF2R action as a tumor suppressor.
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MATERIALS AND METHODS
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Materials
Recombinant human IGF-I and IGF-II were provided by M. H.
Niedenthal (Lilly Research Laboratories, Indianapolis, IN), and IGF-II
was radioiodinated to specific activities between 4085 Ci/g using
carrier-free Na125I (Amersham, arlington Heights, IL) by
Enzymobead reagent (Bio-Rad, Richmond, CA). IGF-II-Sepharose affinity
resin containing 0.5 mg IGF-II per ml resin was prepared as described
previously (47). Disuccinimidyl suberate (DSS) and octyl-ß-glucoside
were from Pierce (Rockford, IL), and BA85 nitrocellulose was from
Schleicher & Schuell (Keene, NH). Antiproteases, bicinchoninic acid
solution, copper sulfate, cyanogen bromide-activated Sepharose 4B,
Man-6-P (disodium salt), BSA, and ovalbumin were obtained from Sigma
Chemical Co. (St. Louis, MO). The anti-FLAG M2 antibody affinity gel,
anti-FLAG M2 antibody, and FLAG peptide were from IBI (New Haven, CT).
Polyclonal antibody to the human c-myc epitope,
MEQKLISEEDLN, was from Upstate Biotechnology (Lake Placid, NY).
Dupont-New England Nuclear (Boston, MA) was the source for
[125I]Protein A. Enhanced chemiluminescence kit (ECL) was
from Amersham Life Sciences. DMEM, trypsin-EDTA, gentamycin, enzymes,
TRIzol reagent, FCS, and antibiotics were acquired from GIBCO-BRL Life
Technologies (Gaithersburg, MD). Deoxynucleoside triphosphates and
Taq polymerase were from Perkin Elmer/Cetus (Norwalk,
CT). Vent polymerase was from New England BioLabs (Beverly, NA),
and the vector pBK-CMV was from Invitrogen (San Diego, CA). M-MLV
reverse transcriptase kit was from Promega (Madison, WI).
Oligonucleotides were synthesized by Integrated DNA Technologies, Inc.
(Coralville, IA), or by the UNMC Molecular Biology Core Facility. 293T
human embryonic kidney cells expressing the SV40 large T antigen were
provided by Dr. Thomas E. Smithgall, University of Nebraska Medical
Center. QIAquick PCR Purification Kit and Plasmid Maxi Kit were from
QIAGEN (Chatsworth, CA). The pCMV5 vector (48) was provided by Dr.
David W. Russell, University of Texas Southwestern Medical Center. The
8.6-kb human IGF2R cDNA (4) was provided by Dr. William S. Sly, St.
Louis University Medical Center.
Preparation of IGF2R Full-Length and Minireceptor Constructs
The pCMV5RIX construct containing the 662-nt 5'-fragment of the
human IGF2R cDNA was prepared as described previously (35). The PCR
with the human IGF2R cDNA serving as the template was employed for
synthesis of six truncated receptor cassettes. Amplification of the
following segments of the receptor cDNA was accomplished by standard
PCR conditions using Taq polymerase (49): repeats 1115 (nt
46757002, encoding residues 15102284); repeats 1113 (nt
46756117, encoding residues 15101981); repeats 1112 (nt
46755541, encoding residues 15101801); repeat 11 (nt 46755100,
encoding residues 15101651) (Fig. 1
). The 5'-primer that was common
to all the constructs was designed to contain an EcoRI
restriction site preceding 18 nt corresponding to the 5'-end of repeat
11 of the human IGF2R cDNA. The 3'-primers were designed to contain 16
nt corresponding to the 3'-end of the indicated segments of the human
IGF2R cDNA followed by the 24-nt sequence encoding the FLAG peptide, a
TGA stop codon, and a BamHI restriction site in the case of
1113 or an XbaI restriction site in the others (Fig. 1A
).
All minireceptor cassettes derived from standard PCR reactions were
digested with EcoRI and BamHI or
XbaI, purified using the QIAquick PCR purification kit,
and then ligated into the linearized pCMV5RIX vector to produce the
truncated receptor-FLAG cDNA constructs illustrated schematically in
Figs. 1
and 5
. Amplification of repeat 13 (nt 55426117, encoding
residues 18021981) was carried out using a 5'-primer containing an
EcoRI site preceding 19 nt corresponding to the 5'-end of
repeat 13 and the same 3'-primer that was used for preparation of the
1113 construct (Fig. 5A
). Amplification of 1113 (
43)
encompassing nt 46756117 with a deletion of nt 58455973 was carried
out as with the 1113 construct except that the template used in the
PCR reaction was a full-length human IGF2R cDNA bearing the deletion of
the 43-residue insert of repeat 13. The receptor cDNA template with
this deletion was prepared by an inverse PCR reaction using as template
a receptor subclone containing a 3.9-kb
HindIII-KpnI fragment encompassing nt 39377847
in pBluescript SK+. The antisense primer contained a
KpnI site followed by 19 nt complementary to the sequence
starting at nt 5844 of the IGF2R cDNA, whereas the sense primer
contained a KpnI site followed by a 19-nt sequence beginning
with nt 5973 of the IGF2R cDNA. The inverse PCR product was subjected
to a partial KpnI digestion and ligated to produce a
construct that encoded a Gly-Thr dipeptide in place of the 43-residue
insertion in repeat 13. The full-length receptor cDNA was reassembled
by stepwise ligation into pGEM-2 followed by subcloning into pCMV5. A
second (
43)IGF2R construct lacking the Gly-Thr dipeptide but bearing
a COOH-terminal c-myc epitope tag was prepared as follows.
The 5,157 nt fragment from nt 162 to nt 5319 was removed by digesting
the IGF2R cDNA with EagI followed by religation. This
smaller insert allowed for addition of sequence encoding the human
c-myc epitope, MEQKLISEEDLN, followed by two stop codons by
amplification with Vent and two primers. The 5'-primer contained an
XhoI site preceding sequence corresponding to nt 94113 of
the receptor cDNA, and the 3'-primer represents sequence complementary
to nt 76027620 at the COOH terminus of the receptor cDNA followed by
27 nt encoding the c-myc epitope, two stop codons, and an
XbaI site. The EagI fragment was subcloned back
into this construct, reconstituting a complete WT c-myc
epitope-tagged receptor construct, IGF2R-myc, which was prepared both
in pCMV5 and pBKCMV. A (
43)IGF2R-myc mutant construct encoding a
complete deletion of the 43-residue type II repeat was prepared by
QuikChange mutagenesis of a receptor cDNA construct encompassing the
region between two PflMI sites (nt 38476315) followed by a
two-step subcloning of the fragment into the IGF2R-myc construct in
pBKCMV. All DNA fragments that had passed through a single-stranded
intermediate and the restriction endonuclease fusion points in each
construct were verified by DNA sequence analysis, conducted by the UNMC
Molecular Biology Core Facility.
Transient Expression of the IGF2R Minireceptors in 293T Cells
For transient expression of receptor constructs, 293T cells were
cultured in DMEM supplemented with 5% FCS plus 50 µg/ml gentamycin
at 37 C in 5% CO2-95% air. Cells were grown to about
7080% confluence in 100-mm dishes. The transfection of plasmids
pCMV5 or pCMV5RIX bearing various truncated IGF2R cDNA constructs was
carried out by a modification of the calcium phosphate precipitation
method described previously (50). The main procedural changes were that
the medium was supplemented with 50 µg/ml gentamycin plus 5% FCS,
and that chloroquine enhancement was not done. The day after
transfection the cells were fed with fresh medium.
Preparation of Lysates and Plasma Membranes from Transfected
Cells
On the sixth day after transfection, cell lysates were prepared
by solubilizing cell monolayers for l h at 4 C in 10 mM
HEPES, pH 7.4, 1 mM MgCl2, 1% (vol/vol) Triton
X-100, plus antiproteases: phenylmethylsulfonyl fluoride (1
µM), aprotinin (20 µg/ml), antipain (10 µg/ml),
benzamidine (80 µg/ml), and leupeptin (10 µg/ml). The suspension
was centrifuged for 10 min at 1200 x g at 4 C, and the
resulting supernatant fractions (lysates) were collected and stored at
-20 C or -80 C. Plasma membranes were isolated on the sixth day after
transfection as described previously (51). Protein concentrations in
the lysates and plasma membrane suspensions were measured using the
bicinchoninic acid assay (Pierce).
Immunoblot Detection of Epitope-Tagged IGF2R Minireceptors and
Full-Length IGF2R
For immunoblotting experiments, aliquots containing 100 µg of
cell lysate protein were heated at 100 C for 7 min in sample buffer
containing 5% SDS and 50 mM dithiothreitol. The samples
were run on 10% or 12% SDS-PAGE and electroblotted to BA85
nitrocellulose. Immunoblots were blocked with 3% nonfat dry milk in
Tris-buffered saline containing 0.1% Tween-20 (milk-TBST) and then
probed with anti-FLAG M2 monoclonal antibody (1:1000 dilution)
according to the manufacturers directions. Detection by ECL was
carried out via biotinylated horseradish peroxidase-streptavidin
complex, using the ECL Western blotting kit. Blots were exposed for
15 min to film.
Immunoblot detection of the full-length, untagged receptors was done
with aliquots containing 200 µg plasma membrane protein, which were
reduced and alkylated under denaturing conditions, and then
electrophoresed on 6% SDS-PAGE as described previously (52). After
electroblotting to BA85 nitrocellulose, the immunoblots were blocked
with milk-TBST and probed with anti-13D antireceptor antibody (1:500
dilution) as described (52). Full-length receptors bearing a human
c-myc epitope tag were electrophoresed on 6% SDS-PAGE after
reduction with 50 mM dithiothreitol and transblotted to BA
85 nitrocellulose. Immunoblots were blocked with milk-TBST, and then
probed with polyclonal anti-myc antibody at 1:1000 dilution.
These blots were developed with [125I]protein A with
detection by autoradiography.
Binding and Affinity Cross-Linking Analysis
Binding and cross-linking of IGF-II to IGF2R minireceptors was
done after immunoadsorption from cell lysates with anti-FLAG M2
affinity gel. Immunoadsorption of the FLAG-tagged proteins was
routinely done by mixing 12 µl of anti-FLAG M2 affinity gel
suspension with cell lysates (50100 µg protein) in buffer
containing 10 mM HEPES, 0.15 M NaCl, 1%
ovalbumin, 0.05% Triton X-100, plus antiproteases on an end-over-end
mixer for 1618 h at 4 C. The washed resins were then incubated with 2
nM [125I]IGF-II with or without unlabeled
IGF-II at the concentrations indicated in the figures, on an
end-over-end mixer overnight at 4 C. Unlabeled IGF-I was included in
these incubations at 100 nM concentration, which completely
blocked 125I-IGF-II binding to IGFBPs; preliminary
experiments revealed that up to 500 nM IGF-I did not
interfere with [125I]IGF-II binding to IGF2R.
Cross-linking was done by incubation with 0.25 mM DSS for
30 min at 3 C. The reaction was quenched by adding 0.8 ml of 0.1
M Tris-HCl, pH 7.4, followed by incubation at 3 C for 15
min (51) and then run either on uniform gels of 6, 7, 10, or 12%
SDS-PAGE. The gels were stained with Coomassie blue, destained, dried,
and then exposed to film. Radioactivity levels in the individual bands
were directly quantified by PhosphorImager analysis (Molecular
Dynamics, Sunnyvale, CA).
IGF-II-Sepharose Affinity-Depletion Assay
An assay for estimating relative binding affinities of
full-length and mutant IGF2Rs was based on their ability to bind IGF-II
immobilized on Sepharose 4B. This assay employed lysates from cells
transfected with c-myc epitope-tagged receptor constructs,
which permitted specific detection of the exogenous IGF2Rs in the
presence of excess endogenous 293T cell IGF2Rs. Thus, the amount of
epitope-tagged receptors remaining in the postresin supernatant after
the IGF-II-Sepharose depletion step was estimated by
anti-myc immunoblot analysis. To compensate for large
differences in expression of WT vs. mutant (
43) IGF2R-myc
constructs in transfected 293T cells, plasma membranes (10 µg
protein) from cells transfected with WT IGF2R-myc were mixed with
membranes from pCMV5 vector-transfected control cells (390 µg
protein) for comparison with membranes (400 µg protein) from cells
transfected with (
43)IGF2R-myc. Dilution of the membranes bearing
the WT IGF2R-myc allowed for appropriate comparison with
(
43)IGF2R-myc membranes because the amounts of total protein and,
more importantly, endogenous 293T cell IGF2Rs were equivalent during
the depletion assay. The membranes were solubilized by incubation with
1% octyl-ß-glucoside in 25 mM HEPES, pH 7.4, 0.15
M NaCl (5 mg protein per ml), at 4 C for 1 h, followed
by centrifugation in a microcentrifuge for 7 min. The supernatant
fractions (extracts) were incubated with 60 µg of packed resin,
either IGF-II-Sepharose (containing 0.5 mg IGF-II per ml resin) or
blank (protein-free) Sepharose, for 16 h at 4 C on an end-over-end
mixer. After centrifugation for 20 sec, the amounts of unbound,
exogenous IGF2Rs present in the supernatant fractions were analyzed by
immunoblotting with the anti-myc antibody. Reductions in
intensity of the anti-myc labeling of the 260-kDa IGF2R
bands from lysates incubated with IGF-II-Sepharose were taken as an
indication of affinity for IGF-II. Although this assay did not permit
direct calculation of affinities, measurement of the percentage of
receptor depleted after exposure to IGF-II-Sepharose, by PhosphorImager
analysis, allowed comparison of relative IGF-II binding affinities. The
blank resin served as a negative control for each sample. The assay was
further validated by showing lack of depletion of a full-length mutant
IGF2R-myc bearing a missense mutation of Ile to Thr at residue 1572
(data not shown). This mutation completely abolishes IGF-II binding to
both minireceptors (35) and full-length receptors (G. R. Devi,
A. T. De Souza, J. C. Byrd, R. L. Jirtle, and R. G.
MacDonald, manuscript in preparation).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Beverly S. Schaffer, P. James Seberger, and
Betty A. Jackson for technical assistance, to Drs. Robert E. Lewis,
Jung H. Y. Park, Oksana Lockridge, David F. Smith, and Thomas E.
Smithgall for helpful advice and discussions during the course of this
work, and Drs. James A. Rogers and Thomas E. Smithgall for
providing 293T cells. We thank Mrs. Margaret H. Niedenthal of Lilly
Research Laboratories for providing IGF-I and IGF-II and Drs. William
S. Sly and David W. Russell for providing cDNAs and vectors.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Richard G. MacDonald, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, Nebraska 68198-4525. E-mail: rgmacdon{at}mail.unmc.edu
This work was supported by NIH Grant DK-44212. DNA sequencing costs
were subsidized by National Cancer Institute Core Grant CA-36727 and
the Nebraska Research Initiative.
Received for publication October 7, 1997.
Revision received July 17, 1998.
Accepted for publication July 28, 1998.
 |
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