©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions (*)

(Received for publication, December 20, 1994; and in revised form, April 7, 1995)

Ryuji Hashimoto (1)(§) Hiroyuki Fujiwara (1) Nobuyuki Higashihashi (1) Tomoko Enjoh-Kimura (1) Hiroaki Terasawa (1) (2) Yoko Fujita-Yamaguchi (3) Fuyuhiko Inagaki (2) James F. Perdue (4) Katsu-ichi Sakano (1)

From the  (1)Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16-13, Kitakasai 1-chome, Edogawa-ku, Tokyo 134, Japan, the (2)Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113, Japan, the (3)Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010, and the (4)Molecular Biology Laboratory, The Jerome H. Holland Laboratory of the American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors remained at 50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to 10% and 3%, respectively, of that of rIGF-II. When the octapeptide including Leu was removed prior to the Cys-Cys intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased to <1% of that of rIGF-II. Substituting Gly for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These results suggest that the side chains of Thr and Leu may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the relative affinity of des-(1-8)- or [Gly]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also <1% of that of rIGF-II, the relative affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II. These results clearly demonstrate that Thr and Leu are important for binding to insulin and IGF-I receptors and Leu is critical for expression of all IGF-II functions.


INTRODUCTION

Insulin-like growth factor (IGF)-I ()and IGF-II are single chain polypeptides of 70 and 67 residues, respectively, that have amino acid sequence homology with each other and with proinsulin(1) . On the basis of this homology, the structure of IGF-I and IGF-II has been divided into four domains designated B, C, A, and D beginning at the N terminus. The B- and A-domains of IGF-II like IGF-I and proinsulin are connected by three intrachain disulfide bonds between Cys-Cys, Cys-Cys, and Cys-Cys that determine the proper folding and receptor binding specificities of the molecule(2, 3) .

Insulin, IGF-I, and IGF-II bind with high affinity to their own specific receptors, i.e. insulin, IGF-I, and IGF-II/CIM6-P receptors. IGF-II, unlike IGF-I and insulin, also binds with moderate to high affinity to the IGF-I and insulin receptors(4, 5, 6) . Because of its high affinity receptor binding properties, our laboratories have undertaken to identify the residues that are critical for IGF-II binding to the insulin, IGF-I, and IGF-II/CIM6-P receptors using site-directed mutagenic procedures. In our initial study, we observed that Tyr, Phe, and Val in the B- and A-domains were essential residues for IGF-II binding to the insulin and IGF-I receptors, whereas Phe, Arg, Ser, Ala, and Leu in the A-domain are residues that were required for its binding to IGF-II/CIM6-P receptors(6) . Using these mutants, we showed evidence that IGF-II stimulated DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells via the IGF-I receptor(6) . Finally in a more recent study, we established that residues Phe, Arg, and Ser were essential for the binding of IGF-II to IGF-binding proteins (IGFBPs) 1-6(7) .

The importance of the N-terminal pentapeptide of IGF-I for their functions has been reported(8) . A naturally occurring truncated form of IGF-I, des-(1-3)-IGF-I, with enhanced mitogenic activity is present in human fetal and adult brain(9, 10) , bovine colostrum(11) , and porcine uterus(12) . The increase in mitogenic potency was postulated to be the result of decreases in the affinity of des-(1-3)-rIGF-I for IGFBPs with the consequence that more unbound growth factor would be available to interact with IGF-I receptors(13, 14) . To date, the isolation of a naturally occurring truncated form of IGF-II has not been reported. However, several deletion mutants of the N-terminal sequence of rIGF-II have been prepared by recombinant procedures, i.e. des-(1-6)- and des-(1-8)-rIGF-II(15) . It was shown that des-(1-6)-rIGF-II had approximately 10-fold reduced affinity for IGFBP-3 but retained an equal affinity for the IGF-I receptor, whereas the relative affinity of des-(1-8)-rIGF-II for IGF receptors and IGFBP-3 was 20% of that of rIGF-II. Our preliminary observations with des-(1-8)-rIGF-II and [Gly]rIGF-II indicated, however, that the relative affinity of both mutants for IGF and insulin receptors are <1% of that of rIGF-II(16) . The solution structure of rIGF-II that recently was determined by us (17) revealed that the N-terminal Ala-Glu region is not well defined, i.e. the B-domain N terminus is flexible because it lacks distance constraints. In this study, we prepared a series of deletion mutants and a substitution mutant in the N-terminal first eight amino acids to more precisely define the contribution this region of the B-domain makes to the binding of rIGF-II to insulin, IGF-I, and IGF-II/CIM6-P receptors.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and Klenow large fragment were purchased from Toyobo (Tokyo, Japan). Des-(1-6)-rIGF-II was purchased from Gropep Pty. Ltd. (Adelaide, Australia). I-human rIGF-I (2,000 Ci/mmol) and I-human rIGF-II (2,000 Ci/mmol) were purchased from Amersham Co. (Buckinghamshire, United Kingdom) with the exception of I-human rIGF-II (2,000 Ci/mmol) that was used to measure the affinity of binding of IGF-II to bovine liver IGF-II/CIM6-P receptors (see below). This material was prepared by the Daiichi Radioisotope Laboratory (Tokyo, Japan). I-Human insulin (2,000 Ci/mmol) and [methyl-H]thymidine (20 Ci/mmol) were from DuPont NEN. Pepsin was purchased from Sigma. All other chemicals were of the highest quality commercially available.

Insulin and IGF-I receptors were purified to apparent homogeneity from Triton X-100-solubilized human placental membrane as described previously(18, 19) . The IGF-II/CIM6-P receptor was partially purified from -octyl glucoside-solubilized rat placenta as described previously(20) . The IGF-II/CIM6-P receptor was purified from bovine liver according to the previously published procedure (21) with some modifications. Briefly, bovine liver acetone powder was prepared from fresh bovine liver. The acetone powder was solubilized with 2% (w/v) -octyl glucoside, followed by wheat germ agglutinin-agarose, rIGF-II-coupled affinity resin, and [Leu]rIGF-II()-coupled affinity resin column chromatography. The purity of the IGF-II/CIM6-P receptor preparation was confirmed by SDS-polyacrylamide gel electrophoresis followed by silver staining (data not shown).

Oligonucleotides were synthesized by an Applied Biosystems model 380A synthesizer, purified as described previously(22) , and sequences confirmed by the dideoxynucleotide chain termination method(23) .

Construction of Mutant rIGF-II Genes for Expression in Escherichia coli

The recombinant human IGF-II gene that was used in this study was synthesized from the known sequence of human IGF-II cDNA as described previously(22) . The N-terminal amino acid substitution or deletion mutants of rIGF-II were constructed by standard site-directed mutagenesis procedures using the synthetic oligonucleotides that are illustrated in Fig. 1. The substitution mutant [Gly]rIGF-II was prepared based on the results of a study (24) that found that the mutant where Leu was replaced by Gly decreased its affinity to the insulin receptor markedly. In the preparation of the N-terminal deletion mutants, des-(1-5)-, des-(1-7)-, and des-(1-8)-rIGF-II, methionine was introduced at N-terminal residues 4, 6, or 7, so that their respective N termini can be removed by cleavage with cyanogen bromide (CNBr) during purification as described below.


Figure 1: DNA sequence of the 5` region and the N-terminal amino acid sequence of human IGF-II. The amino acid of each rIGF-II mutant that has been changed is shown in bold letters. The synthetic oligonucleotides that were used to construct the mutant IGF-II genes are also indicated.



Expression, Preparation, and Chemical Characterization of Recombinant IGF-II Mutants

rIGF-II mutants were expressed in E. coli MC1061 as fusion proteins with rat interleukin-1 (6) . Purified preparations of these fusion proteins were cleaved by CNBr treatment. The IGF-II mutant proteins were refolded, intrachain disulfide bonds formed, and the native molecules were purified by reverse phase-high performance liquid chromatography (rp-HPLC) as described previously(6) .

Proper folding of the purified rIGF-II mutants except for [Gly]rIGF-II was determined by a previously described peptide mapping procedures that involved pepsin digestion, rp-HPLC, and amino acid composition analysis and N-terminal amino acid sequence analysis of the isolated peptide fragments(2, 3, 6) . [Gly]rIGF-II could not be characterized in the same way because the amino acid, i.e. Leu, which is usually cleaved by pepsin becomes more resistant. Therefore, peptides corresponding to those generated when [Gly]rIGF-II was digested were synthesized by an ABI model 431A peptide synthesizer using the selective S-S formation procedures(24) . These were then compared with those obtained when [Gly]rIGF-II was digested for their retention time during analytical rp-HPLC (data not shown).

NMR Measurements

Wild type rIGF-II and [Gly]rIGF-II at concentrations of 2.4 mM in 85% HO, 10% DO, 5% CDCOD, and des-(1-7)- and des-(1-8)-rIGF-II at 0.6 mM in the same solvent were adjusted to a pH of 2.6, and their H NMR spectra were recorded on a UNITY plus 600 spectrometer operating at H 600 MHz and 50 °C with a spectral width of 7,000 Hz. Chemical shifts were referenced relative to the internal standard 2,2-dimethyl-2-silapentane-5-sulfonate.

Characterization of Binding of rIGF-II Mutants to Insulin, IGF-I, and IGF-II/CIM6-P Receptors

Competition for the binding of I-insulin and I-IGF-I to insulin and IGF-I receptors by insulin, IGF-I, rIGF-II, and rIGF-II mutants was carried out using human placental receptors that were purified as described previously(18, 19) . Briefly, duplicate aliquots of 12-32 fmol of receptor were incubated at 4 °C in 0.3 ml of 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% (w/v) bovine serum albumin, 0.075% (v/v) Triton X-100, 20,000 cpm of I-insulin or I-IGF-I, and competing ligands at concentrations of 0.04-1.106 nM. Following an overnight incubation at 4 °C, ligand-receptor complexes were precipitated by the addition of 0.1 ml of 0.4% (w/v) -globulin and 0.5 ml of 25% (w/v) polyethylene glycol. Pellets were washed once and counted in a Pharmacia model 1272 Clini Gamma Counter.

The affinities of the mutants for IGF-II/CIM6-P receptor were measured by using partially purified rat placental membrane-derived receptors prepared as described previously(20) . Briefly, 1.0-1.5 µg of partially purified receptor preparations were incubated in a final volume of 0.4 ml of 100 mM HEPES buffer (pH 8.0) containing 120 mM NaCl, 1 mM EDTA, 1.2 mM MgSO, 2.5 mM KCl, 10 mM glucose, 0.5% (w/v) -octyl glucoside, 0.1% (w/v) bovine serum albumin, 20,000 cpm of I-IGF-II, and competing ligands at concentrations of 0.0075-200 nM. The affinities of the mutants for this receptor were also measured using purified bovine liver IGF-II/CIM6-P receptors. Purified bovine receptors (0.15 µg) were incubated in a final volume of 0.3 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 0.1% (w/v) bovine serum albumin, 0.2% (w/v) -octyl glucoside, 1 mM phenylmethanesulfonyl fluoride, 20,000 cpm of I-IGF-II, and competing ligands at concentrations of 0.004-32 nM. Receptor-ligand complexes were separated from unbound ligands by the polyethylene glycol procedure. Pellets were washed once and counted.

The data from two to four experiments were plotted as the B/B where B is the quantity of I-labeled ligand bound in the presence of a given concentration of competing ligand, and B is the quantity of I-labeled ligand bound in the absence of unlabeled ligand. The apparent equilibrium dissociation constants, K, for the binding of these ligands to the receptors were determined from the nanomoler concentrations of ligand that displaced 50% of specifically bound ligand.

Characterization of the Biological Activities of rIGF-II Mutants

The biological activities of rIGF-II mutants were measured by the incorporation of [H]thymidine into DNA as described previously(6, 25) . In brief, subconfluent cultures of Balb/c 3T3 cells that were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum were plated in 96-well microtiter plates (collagen-coated cell wells; Corning) at a density of 1 10 cells/well. rIGF-II or rIGF-II mutants at concentrations designated in Fig. 5were added to the cells in Dulbecco's modified Eagle's medium for 24 h at 37 °C. At the end of this incubation, the cells were incubated at 37 °C for 2 h with 0.5 µCi/well of [H]thymidine and unincorporated radioactivity removed by washing. The magnitude of [H]thymidine incorporation into cellular DNA was quantified by liquid scintillation counting.


Figure 5: Stimulation of DNA synthesis in Balb/c 3T3 cells by rIGF-II and the five rIGF-II mutants. , rIGF-II; , [Gly]rIGF-II; , des-(1-5)-rIGF-II; , des-(1-6)-rIGF-II; , des-(1-7)-rIGF-II; and , des-(1-8)-rIGF-II. The data are expressed as the mean ± S.D. of four determinations.




RESULTS

Preparation of rIGF-II Mutants

The N-terminal deletion mutants, des-(1-5)-, des-(1-7)-, and des-(1-8)-rIGF-II, and the amino acid substitution mutant [Gly]rIGF-II were prepared as described under ``Experimental Procedures'' and their purity established by SDS-polyacrylamide gel electrophoresis, analytical rp-HPLC, and amino acid composition analysis (data not shown). The position of the three intrachain disulfide bonds between Cys and Cys, Cys and Cys, and Cys and Cys in the three deletion mutants were confirmed by peptide mapping procedures and the [Gly]rIGF-II mutant was found to be properly folded. The N-terminal amino acids for des-(1-5)-, des-(1-7)-, and des-(1-8)-rIGF-II were determined by protein sequencing to be Glu, Leu, and Cys, respectively. The composition of the three disulfide bonds and mutation sites of these mutants are illustrated in Fig. 2. One-dimensional NMR spectra of des-(1-7)-, des-(1-8)-, and [Gly]rIGF-II mutants were compared together with that of the wild type rIGF-II in Fig. 3. The spectra were similar with each other, supporting that three-dimensional structure of the mutants were not significantly different from that of the wild type rIGF-II.


Figure 2: Schematic illustration of human IGF-II and the substitutions introduced into the five rIGF-II mutants. The three disulfide bonds are connected by lines and the positions of the cysteine residues are numbered.




Figure 3: Comparison of H NMR spectrum between rIGF-II and rIGF-II mutants. Amide and aromatic proton regions of 600 MHz H NMR spectra of rIGF-II (A), des-(1-7)-rIGF-II (B), des-(1-8) rIGF-II (C), and [Gly]rIGF-II (D) were measured in 85% HO, 10% DO, 5% CDCOD at 50 °C as described under ``Experimental Procedures''.



Relative Affinity of rIGF-II Mutants for Binding to Insulin, IGF-I, and IGF-II/CIM6-P Receptors

The K values for the binding of insulin and rIGF-II to human placental membrane insulin receptors were 0.12 and 0.9 nM, respectively (Fig. 4A). The K values for the binding of rIGF-I and rIGF-II to human IGF-I receptors were 0.12 and 0.33 nM, respectively (Fig. 4B). These values are similar to those observed in an earlier study (6) for these ligands. The K of 0.1-0.4 nM that was calculated for rIGF-II binding to the rat placental IGF-II/CIM6-P receptors (Fig. 4C) is 3-10 times higher than was observed in our initial study in which the K was 0.04 nM(6) . This difference probably reflects the use of two to three times more of the partially purified receptors than was used previously in order to obtain the same percent of binding. A relative affinity of binding of each mutant to the three receptors was calculated from their displacement curves shown in Fig. 4and compared with that of rIGF-II, which was set to 100% (Table 1).


Figure 4: Characterization of the binding of rIGF-II mutants to insulin, IGF-I, and IGF-II/CIM6-P receptors. Competitive inhibition of I-human IGF-II binding to purified human placental insulin receptors (A), purified human placental IGF-I receptors (B), and partially purified rat placental IGF-II/CIM6-P receptors (C) by insulin (⊡), IGF-I (), rIGF-II (), [Gly]rIGF-II (), des-(1-5)-rIGF-II (), des-(1-6)-rIGF-II (), des-(1-7)-rIGF-II (); and des-(1-8)-rIGF-II (). The results are the mean of two to four experiments and are plotted as B/B, where B is the quantity of I-labeled ligand bound in the presence of a given concentration of competing ligand and B is the quantity of I-labeled ligand bound in its absence.





The deletion of the first five, then six, and finally seven amino acids from the N-terminal end of the B-domain caused, with one exception, i.e. the IGF-I receptor data of des-(1-6)-rIGF-II, a progressive decrease in the relative affinity of the rIGF-II mutants for the insulin and IGF-I receptors (Fig. 4, A and B). With seven amino acids removed, des-(1-7)-rIGF-II bound to the insulin and IGF-I receptors with 11 and 3.3%, respectively, of the affinity of rIGF-II (Table 1). In sharp contrast, the sequential deletion, i.e. residues 5 to 7, had the opposite effect on the binding of the IGF-II mutants to the IGF-II/CIM6-P receptor. For example, the relative affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for the rat placental membrane IGF-II/CIM6-P receptor were 350, 700, and 1,200% of that of rIGF-II. This unexpected result prompted us to examine the effect of N-terminal amino acid deletions of IGF-II on its binding to bovine liver IGF receptors. In this study, the K for the binding of rIGF-II to bovine liver IGF-II/CIM6-P receptors was 0.06 nM. This K value was lower than that of the rat placental IGF-II/CIM6-P receptor preparation used in the current study but was similar to the value obtained in the previous study(6) . The affinities of des-(1-5)- and des-(1-7)-rIGF-II for the bovine receptors were 300 and 200%, respectively, of that of rIGF-II (Table 1). The affinity of the latter for the bovine receptor was much less than it was for the rat placental IGF-II/CIM6-P receptor, i.e. for 1,200%. However, the affinity of des-(1-5)-rIGF-II was about the same for both sources of IGF-II/CIM6-P receptor. Thus, the results of binding of the IGF-II mutants to both rat placental and bovine liver IGF-II/CIM6-P receptors indicated that deletion of the first seven N-terminal amino acids increased the affinity of the IGF-II mutants for the IGF-II/CIM6-P receptors.

The greatest adverse effect on the binding of the rIGF-II to all three receptors occurred when Gly was substituted for Leu or the N-terminal sequence was deleted to Cys. The binding of [Gly]rIGF-II and des-(1-8)-rIGF-II was <2.0% of that of rIGF-II. In these analyses, [Gly]rIGF-II tended to have relatively higher affinities for all three receptors when compared with des-(1-8)-rIGF-II. The effect of a single amino acid removal from IGF-II on its ability to bind to the IGF-II/CIM6-P receptors was clearly evident when deleting Leu from des-(1-7)-rIGF-II decreased the binding affinity of the new mutant by 3 logs. Finally, this result could not be due to drastically altered three-dimensional structure of the mutants, since the overall structure and proper disulfide bond formation of [Gly]rIGF-II or des-(1-8)-rIGF-II have been confirmed by one-dimensional NMR and peptide mapping analysis, respectively.

Characterization of the Biological Activities of rIGF-II Mutants

[H]Thymidine incorporation into the DNA of Balb/c 3T3 cells was used in our previous study to show that the amino acids Phe, Tyr, and Val that are required for the binding of IGF-II to IGF-I receptors were also required for the stimulation of DNA synthesis(6) . Using the same approach, the magnitude of incorporation of [H]thymidine into Balb/c 3T3 cells treated with varying concentrations of the N-terminal amino acid deletion mutants of rIGF-II decreased as the number of the amino acids deleted increased. For example, des-(1-5)-rIGF-II was equipotent with rIGF-II and des-(1-6)- and des-(1-7)-rIGF-II were proportionally less potent (Fig. 5). Des-(1-8)- and [Gly]rIGF-II at concentrations of 200 nM, that produced a near-maximum response with rIGF-II, were 10-fold less potent as stimulators of DNA synthesis. Among the three receptors tested (Table 1), the relationship between mitogenic potency and N-terminal amino acid length to Thr of the deletion mutants was correlated the best with their ability to bind to IGF-I receptors.


DISCUSSION

In our previous studies with rIGF-II mutants, it was established that Phe, Tyr, and Val were essential for IGF-II binding to insulin and IGF-I receptors and that Phe, Arg, Ser, Ala, and Leu were essential for its interaction with IGF-II/CIM6-P receptors(6, 16) . Furthermore, Phe, Arg, and Ser were also involved in IGF-II binding to IGFBPs 1-6 (7, 16) . The positions of these residues in the IGF-II molecule were determined by NMR analysis which showed that these residues lay on the surface of the molecule(17) . In the present study, the contribution of the first eight N-terminal amino acids, i.e. Ala, Tyr, Arg, Pro, Ser, Glu, Thr, and Leu, of IGF-II on its binding affinity for IGF and insulin receptors were examined.

Beginning with des-(1-5)-rIGF-II, the relative affinity of the N-terminal amino acid deletion mutants of rIGF-II for the IGF-I and insulin receptors decreased as this octapeptide decreased in length (Fig. 4, A and B, and Table 1). When the eight amino acids were removed, the relative affinity of the des-(1-8) mutant was <1% of that of rIGF-II. The decrease in the relative affinity of the deletion mutants for the IGF-I receptor with a decrease in the length of their N-terminal octapeptide sequence was paralleled by the decrease in their ability to stimulate thymidine incorporation into cultured Balb/c 3T3 cells (Fig. 5). These results are similar in general to what was observed by Lthi et al.(15) in studies of the binding of des-(1-6)- and des-(1-8)-rIGF-II to IGF-I receptors and their biological activities on NIH 3T3 cells. For the comparison, a summary of relative affinities for analogous mutants of insulin and IGF-I from other laboratories is tabulated (Table 2). According to Lthi et al.(15) , the relative affinity of des-(1-8)-rIGF-II for IGF receptors was reduced to 20% of that of rIGF-II. This result is different from our observations that the same mutant has relative affinities for IGF and insulin receptors that are <1% of that of rIGF-II. It could be due to the sources of the receptors used for the different analyses. Our results with des-(1-8)- and [Gly]rIGF-II are instead very similar to those observed with the analogous corresponding insulin mutants, des-(1-6)- and [Gly]insulin (24) as well as a series of IGF-I deletion mutants (Table 2). We did not test additional shorter or longer deletion mutants, since in the first case, des-(1-5)-rIGF-II bound to IGF-I receptors with an affinity that was similar to rIGF-II (Table 1). While the deletion of the next amino acid, i.e. Cys, would eliminate the disulfide bond between it and Cys.



Des-(1-7)-rIGF-II bound to the IGF-I and insulin receptors with 3 and 11% of the relative affinity, respectively, of that of rIGF-II. The relative affinity of des-(1-7)-rIGF-II for a partially purified preparation of rat placental IGF-II/CIM6-P receptors, however, was increased 12-fold (Fig. 4C and Table 1). Des-(1-5)- and des-(1-6)-rIGF-II also showed increased affinity for the IGF-II/CIM6-P receptors. These observations were generally confirmed in competitive ligand binding studies with purified bovine liver IGF-II/CIM6-P receptors and demonstrate the pivotal role that the first seven amino acids in IGF-II serve in defining its specificity for IGF-I and insulin receptors versus the IGF-II/CIM6-P receptors.

Three-dimensional structural analysis of IGF-II by NMR that was carried out recently by us (17) showed that Thr is located approximate to the A-domain residues of Phe and Ser and the disulfide bridge of Cys-Cys (Fig. 6). Phe and Ser are part of the binding site that interacts with the IGF-II/CIM6-P receptors (6) and the IGFBPs 1-6 (7) . The interaction between Thr and Phe/Ser and Cys-Cys may serve to stabilize the apparently flexible N-terminal hexapeptide of IGF-II that is involved in the binding of the growth factor to the IGFBPs and insulin and IGF-I receptors. However, the apparent greater affinity with which des-(1-7)-rIGF-II binds to IGF-II/CIM6-P receptors suggests that the N-terminal amino acid sequence serves to structurally modulate the magnitude of IGF-II binding. Alternatively, the removal of this sequence could result in the establishment of a higher affinity binding site because of the greater ability of the mutant to induce a fit between its more exposed surface residues especially Phe/Ser and the IGF-II/CIM6-P receptor. The resolution of these two possibilities awaits further study.


Figure 6: Three-dimensional structure of rIGF-II. Schematic ribbon drawing of rIGF-II that had been subjected to NMR spectroscopy. Side chains of the amino acids that are important for this study are shown and labeled, Glu in pink, Thr in light green, Phe in purple, Phe in orange, Arg in blue, Ser in green, Ala in red, Leu in light blue, and the three disulfide bonds in yellow. The N and C termini of rIGF-II are labeled as N and C, respectively.



The decrease in affinity of des-(1-8)-rIGF-II for the IGF-I, insulin, and IGF-II/CIM6-P receptors was not the consequence of simply removing the N-terminal octapeptide because [Gly]rIGF-II decreased the affinity to these receptors about the same extent (Table 1). Finally, [Gly]rIGF-II also had relative affinities for IGFBPs 1-6 that were less than 1% of that of rIGF-II(16) . The solution structural analysis of IGF-II revealed that the side chain of Leu was not likely to be involved in a structural stabilization of IGF-II molecule, since nuclear Overhauser effect signals between Leu and other regions were not detected. Nevertheless, Leu and Leu may serve pivotal roles in determining the biological activity of IGF-II and presumably IGF-I, respectively. The evidence to suggest such a role stems from the observation that the dramatic decreases in the binding affinities of des-(1-8)- and [Gly]rIGF-II for the IGF and insulin receptors are results that are very similar to what was observed for des-(1-6)- and [Gly]insulin in binding to the insulin receptor(24) . As evidenced from one-dimensional NMR analysis, the decrease in affinity of des-(1-8)- and [Gly]rIGF-II for the receptors and IGFBPs was not due to the drastic alternation of their three-dimensional structure (Fig. 3). These results strongly suggested that the side chain of Leu of IGF-II are required for its binding to the IGF-I, insulin, and especially IGF-II/CIM6-P receptors.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA47150 (to J. F. P.) and DK34427 (to Y. F.-Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be sent: Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16-13 Kitakasai 1-chome, Edogawa-ku, Tokyo 134, Japan.

The abbreviations used are: IGF, insulin-like growth factor; rIGF-I or -II, recombinant insulin-like growth factor-I or -II; CIM6-P, cation-independent mannose 6-phosphate; CNBr, cyanogen bromide; rp-HPLC, reverse phase-high performance liquid chromatography.

[Leu]rIGF-II binds specifically to IGF-II/CIM6-P receptors but not to insulin and IGF-I receptors (6).


ACKNOWLEDGEMENTS

We express our thanks to Dr. D. Kohda and Dr. H. Hatanaka for many helpful discussions about the tertiary structure of IGF-II. We are grateful to Dr. M. Furusawa for his valuable advice, discussions, and continuous encouragement. We also thank Y. Kaibori, Y. Arai, and K. Sato for their excellent technical assistance.


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