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Molecular Endocrinology 12 (11): 1661-1672
Copyright © 1998 by The Endocrine Society

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 receptor’s 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 IGF2R’s 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 11–13 or 11–15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1–2 nM), whereas constructs containing repeat 11 only or repeats 11–12 did not (IC50 = 10–20 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 11–13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10–20 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1–3 and 7–9 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 receptor’s IGF-II binding properties may be key in tumorigenesis.

The potential role of the IGF2R in human cancer has enhanced interest in understanding the receptor’s 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 8–11 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 10–15 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 12–15 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 IGF2R’s 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 IGF2R’s 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo. 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. 1BGo). Small discrepancies between the estimated Mr for the 11–12 and 11–13 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.

 
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. 2Go 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.

 
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. 3Go). A gradual displacement of [125I]IGF-II labeling of the minireceptor constructs 11, 11–12, 11–13, and 11–15 was observed with increasing concentrations of unlabeled IGF-II (Fig. 3Go). However, complete displacement of the major radioactive band in the case of the 11–13 and 11–15 minireceptors occurred at lower concentrations of unlabeled IGF-II (1–2 nM), when compared with the minireceptors encompassing repeat 11 (10–20 nM) or repeats 11–12 (15–30 nM). The top panel of Fig. 3Go 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.

 
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. 4Go). The curves for minireceptors 11 and 11–12 were shifted to the right relative to those of the full-length receptor and minireceptors 11–13 and 11–15. Calculation of the unlabeled IGF-II concentrations required to achieve 50% displacement of binding (IC50) indicated that minireceptors 11 and 11–12 had IC50 values of 15–20 nM, which were approximately 1 order of magnitude higher than those of minireceptors 11–13, 11–15, and the full-length receptor [IC50 values in the range 1–5 nM (Table 1Go)].



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  		Figure 4. Quantitative Analysis of Radiolabeled 125I-IGF-II Displacement from IGF2R Minireceptors

The gels shown in Fig. 3Go 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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  		Table 1. Summary of IC50 Values for IGF-II Binding to Minireceptors and Full-Length IGF2R

 
Deletion of the Type II Repeat in Repeat 13 Reduces Affinity of the 11–13 Minireceptor
Since the 11–13 and 11–15 minireceptors showed high affinity comparable to the full-length IGF2R and the 11–12 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. 5AGo), using a strategy similar to that described in the legend to Fig. 1Go: minireceptor construct 11–13({Delta}43), which contained repeats 11–13 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. 5BGo). A small discrepancy between the estimated Mr for the 11–13({Delta}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).



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  		Figure 5. Preparation and Expression of the Repeats 11–13({Delta}43) and 13 IGF2R Minireceptors

A, The cDNA cassettes for preparation of repeat 13 and 11–13({Delta}43) minireceptor constructs were synthesized by PCR using as template the human IGF2R receptor cDNA or an IGF2R cDNA bearing a deletion of the unique 43-residue insert in repeat 13, respectively. The other major features of these constructs are the same as described in the legend to Fig. 1AGo. B, Lysates prepared from 293T cells on the sixth day after transfection with the minireceptor constructs were assayed for expression of the IGF2R minireceptors by immunoblotting using the M2 monoclonal antibody coupled to ECL.

 
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. 6Go 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) 11–13 minireceptor, the 11–13({Delta}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 11–13({Delta}43)-IGF-II complex occurred at higher concentrations of unlabeled IGF-II (10–20 nM) relative to that of the 11–13-IGF-II complex (IC50 = 1.5–4 nM, Figs. 3Go and 4Go). 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 11–13({Delta}43) minireceptor relative to the WT 11–13 minireceptor (data not shown).



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  		Figure 6. Affinity Labeling of Minireceptors 11, 13, and 11–13({Delta}43) with [125I]IGF-II

Minireceptors 11–13({Delta}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.

 
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 ({Delta}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 ({Delta}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 ({Delta}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 ({Delta}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 ({Delta}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. 7AGo). The band corresponding to ({Delta}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.



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  		Figure 7. Analysis of IGF-II Binding Characteristics of Full-Length WT and Mutant ({Delta}43) IGF2Rs

A, Immunoblot analysis. Plasma membranes (200 µg protein) prepared from 293T cells on the sixth day after transfection with the WT IGF2R-myc and ({Delta}43)IGF2R-myc constructs were analyzed for expression of the epitope-tagged receptors by immunoblotting with an anti-myc antibody. The arrow indicates the WT IGF2R-myc. Note that the band corresponding to ({Delta}43)IGF2R-myc is of slightly lower Mr than the WT receptor. B, IGF-II-Sepharose affinity depletion analysis. c-myc-tagged receptors solubilized from 293T cell plasma membranes (400 µg protein) were assayed for IGF-II binding capability by incubation with IGF-II-Sepharose or blank Sepharose, as indicated and as described in Materials and Methods. The amount of unbound c-myc-tagged receptors remaining in the postresin supernatants was assayed by immunoblotting with an anti-myc antibody, an autoradiograph of which is shown. The reduction in intensity of the WT IGF2R-myc band upon incubation with IGF-II-Sepharose is indicative of its affinity for IGF-II, whereas ({Delta}43)IGF2R-myc shows little reduction in band intensity after incubation with IGF-II resin, indicating a reduced IGF-II binding affinity.

 
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 {Delta}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. 7BGo). 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 ({Delta}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 ({Delta}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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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 11–13({Delta}43) minireceptor bearing a deletion of the unique 43-residue insert exhibited low IGF-II binding affinity relative to the WT 11–13 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 11–13({Delta}43) minireceptor had very low IGF-II binding affinity relative to the WT 11–13 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 receptor’s 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 {alpha}-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 IGF2R’s 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 40–85 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 11–15 (nt 4675–7002, encoding residues 1510–2284); repeats 11–13 (nt 4675–6117, encoding residues 1510–1981); repeats 11–12 (nt 4675–5541, encoding residues 1510–1801); repeat 11 (nt 4675–5100, encoding residues 1510–1651) (Fig. 1Go). 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 11–13 or an XbaI restriction site in the others (Fig. 1AGo). 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. 1Go and 5Go. Amplification of repeat 13 (nt 5542–6117, encoding residues 1802–1981) 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 11–13 construct (Fig. 5AGo). Amplification of 11–13 ({Delta}43) encompassing nt 4675–6117 with a deletion of nt 5845–5973 was carried out as with the 11–13 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 3937–7847 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 ({Delta}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 94–113 of the receptor cDNA, and the 3'-primer represents sequence complementary to nt 7602–7620 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 ({Delta}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 3847–6315) 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 70–80% 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 manufacturer’s directions. Detection by ECL was carried out via biotinylated horseradish peroxidase-streptavidin complex, using the ECL Western blotting kit. Blots were exposed for 1–5 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 (50–100 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 16–18 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 ({Delta}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 ({Delta}43)IGF2R-myc. Dilution of the membranes bearing the WT IGF2R-myc allowed for appropriate comparison with ({Delta}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.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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