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• W2017979183 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 Gly8 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 Leu8 was removed prior to the Cys9-Cys47 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 Gly8 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 Thr7 and Leu8 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 [Gly8]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 Thr7 and Leu8 are important for binding to insulin and IGF-I receptors and Leu8 is critical for expression of all IGF-II functions. 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 Gly8 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 Leu8 was removed prior to the Cys9-Cys47 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 Gly8 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 Thr7 and Leu8 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 [Gly8]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 Thr7 and Leu8 are important for binding to insulin and IGF-I receptors and Leu8 is critical for expression of all IGF-II functions. Insulin-like growth factor (IGF)1-I 1The abbreviations used are: IGFinsulin-like growth factorrIGF-I or −IIrecombinant insulin-like growth factor-I or −IICIM6-Pcation-independent mannose 6-phosphateCNBrcyanogen bromiderp-HPLCreverse phase-high performance liquid chromatography. 1The abbreviations used are: IGFinsulin-like growth factorrIGF-I or −IIrecombinant insulin-like growth factor-I or −IICIM6-Pcation-independent mannose 6-phosphateCNBrcyanogen bromiderp-HPLCreverse phase-high performance liquid chromatography. 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(1Humbel R.E. Eur. J. Biochem. 1990; 190: 445-462Google Scholar). 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 Cys9-Cys47, Cys21-Cys60, and Cys46-Cys51 that determine the proper folding and receptor binding specificities of the molecule(2Smith M.C. Cook J.A. Furman T.C. Occolowitz J.L. J. Biol. Chem. 1989; 264: 9314-9321Google Scholar, 3Iwai, M Kobayashi, M. Tamura K. Ishii Y. Yamada H. Niwa M. J. Biochem. (Tokyo). 1989; 106: 949-951Google Scholar).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(4Casella S.J. Han V.K. D'Ercole A.J. Svoboda M.E. Van Wyk J.J. J. Biol. Chem. 1986; 261: 9268-9273Google Scholar, 5Bayne M.L. Applebaum J. Chicchi G.G. Miller R.E. Cascieri M.A. J. Biol. Chem. 1990; 265: 15648-15652Google Scholar, 6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). 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 Tyr27, Phe26, and Val43 in the B- and A-domains were essential residues for IGF-II binding to the insulin and IGF-I receptors, whereas Phe48, Arg49, Ser50, Ala54, and Leu55 in the A-domain are residues that were required for its binding to IGF-II/CIM6-P receptors(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). 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(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). Finally in a more recent study, we established that residues Phe48, Arg49, and Ser50 were essential for the binding of IGF-II to IGF-binding proteins (IGFBPs) 1-6(7Bach L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Google Scholar).The importance of the N-terminal pentapeptide of IGF-I for their functions has been reported(8Bagley C.J. May B.L. Szabo L. McNamara P.J. Ross M. Francis G.L. Ballard F.J. Wallace J.C. Biochem. J. 1989; 259: 665-671Google Scholar). 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(9Sara V.R. Carlsson-Skwirut C. Andersson C. Hall E. Sjgren B. Holmgren A. Jrnvall H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4904-4907Google Scholar, 10Carlsson-Skwirut C. Jrnvall H. Holmgren A. Andersson C. Bergman T. Lundquist G. Sjgren B. Sara V.R. FEBS Lett. 1986; 201: 46-50Google Scholar), bovine colostrum(11Francis G.L. Upton F.M. Ballard F.J. McNeil K.A. Wallace J.C. Biochem. J. 1988; 251: 95-103Google Scholar), and porcine uterus(12Ogasawara M. Karey K.P. Marquardt H. Sirbasku D.A. Biochemistry. 1989; 28: 2710-2721Google Scholar). 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(13Cascieri M.A. Hayes N.S. Bayne M.L. J. Cell. Physiol. 1989; 139: 181-188Google Scholar, 14Ross M. Francis G.L. Szabo L. Wallace J.C. Ballard F.J. Biochem. J. 1989; 258: 267-272Google Scholar). 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(15Lthi C. Roth B.V. Humbel R.E. Eur. J. Biochem. 1992; 205: 483-490Google Scholar). 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 [Gly8]rIGF-II indicated, however, that the relative affinity of both mutants for IGF and insulin receptors are <1% of that of rIGF-II(16Perdue J.F. Bach L.A. Hashimoto R. Sakano K. Fujita-Yamaguchi Y. Fujiwara H. Rechler M.M. Baxter R.C. Gluckman P.D. Rosenfeld R.G. The Insulin-like Growth Factors and Their Regulatory Proteins. Elsevier Science Publishing Co., New York1994: 67-76Google Scholar). The solution structure of rIGF-II that recently was determined by us (17Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Google Scholar) revealed that the N-terminal Ala1-Glu6 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 PROCEDURESMaterialsRestriction endonucleases and Klenow large fragment were purchased from Toyobo (Tokyo, Japan). Des-(1-6)-rIGF-II was purchased from Gropep Pty. Ltd. (Adelaide, Australia). 125I-human rIGF-I (2,000 Ci/mmol) and 125I-human rIGF-II (2,000 Ci/mmol) were purchased from Amersham Co. (Buckinghamshire, United Kingdom) with the exception of 125I-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). 125I-Human insulin (2,000 Ci/mmol) and [methyl-3H]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(18Fujita-Yamaguchi Y. Choi S. Sakamoto Y. Itakura K. J. Biol. Chem. 1983; 258: 5045-5049Google Scholar, 19LeBon T.R. Jacobs S. Cuatrecasas P. Kathuria S. Fujita-Yamaguchi Y. J. Biol. Chem. 1986; 261: 7685-7689Google Scholar). The IGF-II/CIM6-P receptor was partially purified from β-octyl glucoside-solubilized rat placenta as described previously(20Perdue J.F. Chan J.K. Thibault C. Radaj P. Mills B. Daughaday W.H. J. Biol. Chem. 1983; 258: 7800-7811Google Scholar). The IGF-II/CIM6-P receptor was purified from bovine liver according to the previously published procedure (21Sahagian G.G. Distler J.J. Jourdian G.W. Methods Enzymol. 1982; 83: 392-396Google Scholar) with some modifications. Briefly, bovine liver acetone powder was prepared from fresh bovine liver. The acetone powder was solubilized with 2% (w/v) β-octyl glucoside, followed by wheat germ agglutinin-agarose, rIGF-II-coupled affinity resin, and [Leu27]rIGF-II2 2[Leu27]rIGF-II binds specifically to IGF-II/CIM6-P receptors but not to insulin and IGF-I receptors (6). -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(22Marumoto Y. Sato Y. Fujiwara H. Sakano K. Saeki Y. Agata M. Furusawa M. Maeda S. J. Gen. Virol. 1987; 68: 2599-2606Google Scholar), and sequences confirmed by the dideoxynucleotide chain termination method(23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar).Construction of Mutant rIGF-II Genes for Expression in Escherichia coliThe 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(22Marumoto Y. Sato Y. Fujiwara H. Sakano K. Saeki Y. Agata M. Furusawa M. Maeda S. J. Gen. Virol. 1987; 68: 2599-2606Google Scholar). 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 [Gly8]rIGF-II was prepared based on the results of a study (24Nakagawa S.H. Tager H.S. J. Biol. Chem. 1991; 266: 11502-11509Google Scholar) that found that the mutant where LeuB6 was replaced by GlyB6 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.Expression, Preparation, and Chemical Characterization of Recombinant IGF-II MutantsrIGF-II mutants were expressed in E. coli MC1061 as fusion proteins with rat interleukin-1 (6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). 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(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar).Proper folding of the purified rIGF-II mutants except for [Gly8]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(2Smith M.C. Cook J.A. Furman T.C. Occolowitz J.L. J. Biol. Chem. 1989; 264: 9314-9321Google Scholar, 3Iwai, M Kobayashi, M. Tamura K. Ishii Y. Yamada H. Niwa M. J. Biochem. (Tokyo). 1989; 106: 949-951Google Scholar, 6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). [Gly8]rIGF-II could not be characterized in the same way because the amino acid, i.e. Leu8, which is usually cleaved by pepsin becomes more resistant. Therefore, peptides corresponding to those generated when [Gly8]rIGF-II was digested were synthesized by an ABI model 431A peptide synthesizer using the selective S-S formation procedures(24Nakagawa S.H. Tager H.S. J. Biol. Chem. 1991; 266: 11502-11509Google Scholar). These were then compared with those obtained when [Gly8]rIGF-II was digested for their retention time during analytical rp-HPLC (data not shown).NMR MeasurementsWild type rIGF-II and [Gly8]rIGF-II at concentrations of 2.4 mM in 85% H2O, 10% D2O, 5% CD3CO2D, 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 1H NMR spectra were recorded on a UNITY plus 600 spectrometer operating at 1H 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 ReceptorsCompetition for the binding of 125I-insulin and 125I-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(18Fujita-Yamaguchi Y. Choi S. Sakamoto Y. Itakura K. J. Biol. Chem. 1983; 258: 5045-5049Google Scholar, 19LeBon T.R. Jacobs S. Cuatrecasas P. Kathuria S. Fujita-Yamaguchi Y. J. Biol. Chem. 1986; 261: 7685-7689Google Scholar). 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 125I-insulin or 125I-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) γ-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(20Perdue J.F. Chan J.K. Thibault C. Radaj P. Mills B. Daughaday W.H. J. Biol. Chem. 1983; 258: 7800-7811Google Scholar). 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 MgSO4, 2.5 mM KCl, 10 mM glucose, 0.5% (w/v) β-octyl glucoside, 0.1% (w/v) bovine serum albumin, 20,000 cpm of 125I-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) β-octyl glucoside, 1 mM phenylmethanesulfonyl fluoride, 20,000 cpm of 125I-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/Bo where B is the quantity of 125I-labeled ligand bound in the presence of a given concentration of competing ligand, and Bo is the quantity of 125I-labeled ligand bound in the absence of unlabeled ligand. The apparent equilibrium dissociation constants, Kd(app), 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 MutantsThe biological activities of rIGF-II mutants were measured by the incorporation of [3H]thymidine into DNA as described previously(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar, 25Saito Y. Yamada H. Niwa M. Ueda I. J. Biochem. (Tokyo). 1987; 101: 123-134Google Scholar). 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 × 104 cells/well. rIGF-II or rIGF-II mutants at concentrations designated in Fig. 5 were 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 [3H]thymidine and unincorporated radioactivity removed by washing. The magnitude of [3H]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; □, [Gly8]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.View Large Image Figure ViewerDownload (PPT)RESULTSPreparation of rIGF-II MutantsThe N-terminal deletion mutants, des-(1-5)-, des-(1-7)-, and des-(1-8)-rIGF-II, and the amino acid substitution mutant [Gly8]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 Cys9 and Cys47, Cys21 and Cys60, and Cys46 and Cys51 in the three deletion mutants were confirmed by peptide mapping procedures and the [Gly8]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 Glu6, Leu8, and Cys9, 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 [Gly8]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.View Large Image Figure ViewerDownload (PPT)Figure 3:Comparison of 1H NMR spectrum between rIGF-II and rIGF-II mutants. Amide and aromatic proton regions of 600 MHz 1H NMR spectra of rIGF-II (A), des-(1-7)-rIGF-II (B), des-(1-8) rIGF-II (C), and [Gly8]rIGF-II (D) were measured in 85% H2O, 10% D2O, 5% CD3CO2D at 50°C as described under “Experimental Procedures”.View Large Image Figure ViewerDownload (PPT)Relative Affinity of rIGF-II Mutants for Binding to Insulin, IGF-I, and IGF-II/CIM6-P ReceptorsThe Kd(app) 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 Kd(app) 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 (6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar) for these ligands. The Kd(app) 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 Kd(app) was 0.04 nM(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). 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. 4 and 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 125I-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 (•), [Gly8]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/Bo, where B is the quantity of 125-labeled ligand bound in the presence of a given concentration of competing ligand and Bo is the quantity of 125I-labeled ligand bound in its absence.View Large Image Figure ViewerDownload (PPT)TABLE I Open table in a new tab 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 Kd(app) for the binding of rIGF-II to bovine liver IGF-II/CIM6-P receptors was 0.06 nM. This Kd(app) 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(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). 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 Leu8 or the N-terminal sequence was deleted to Cys9. The binding of [Gly8]rIGF-II and des-(1-8)-rIGF-II was <2.0% of that of rIGF-II. In these analyses, [Gly8]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 Leu8 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 [Gly8]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[3H]Thymidine incorporation into the DNA of Balb/c 3T3 cells was used in our previous study to show that the amino acids Phe26, Tyr27, and Val43 that are required for the binding of IGF-II to IGF-I receptors were also required for the stimulation of DNA synthesis(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar). Using the same approach, the magnitude of incorporation of [3H]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 [Gly8]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 Thr7 of the deletion mutants was correlated the best with their ability to bind to IGF-I receptors.DISCUSSIONIn our previous studies with rIGF-II mutants, it was established that Phe26, Tyr27, and Val43 were essential for IGF-II binding to insulin and IGF-I receptors and that Phe48, Arg49, Ser50, Ala54, and Leu55 were essential for its interaction with IGF-II/CIM6-P receptors(6Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Google Scholar, 16Perdue J.F. Bach L.A. Hashimoto R. Sakano K. Fujita-Yamaguchi Y. Fujiwara H. Rechler M.M. Baxter R.C. Gluckman P.D. Rosenfeld R.G. The Insulin-like Growth Factors and Their Regulatory Proteins. Elsevier Science Publishing Co., New York1994: 67-76Google Scholar). Furthermore, Phe48, Arg49, and Ser50 were also" @default.
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• W2017979183 title "N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr7 and Leu8 Important for Binding to Insulin and IGF-I Receptors and Leu8 Critical for All IGF-II Functions" @default.
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