Binding Sites and Binding Properties of Binary and Ternary Complexes of Insulin-like Growth Factor-II (IGF-II), IGF-binding Protein-3, and Acid-labile Subunit

Abstract

We have examined regions of rat IGF-binding protein-3 (IGFBP-3) important for complex formations using two kinds of deletion mutants, three kinds of chimera molecules between rat IGFBP-3 and rat IGFBP-2, and a synthetic peptide (41 residues, Glu52-Ala92) derived from rat IGFBP-3. Solid-phase binding assays using 96-well microtiter plates were designed to quantitate the relative binding affinities. It was found that not only the IGFBP-3 derivatives with the amino-terminal, cysteine-rich domain (N domain) but also the synthetic peptide maintained affinity for IGF-II. Ternary complex formation was observed with full-length IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domain (C domain) of which was derived from IGFBP-3, unlike the mutants lacking the C domain and the chimera IGFBPs, the C domain of which was derived from IGFBP-2. These results were confirmed by affinity cross-linking experiments. Furthermore, the IGFBP-3 derivatives that possessed the C domain of IGFBP-3 bound to the acid-labile subunit, even in the absence of IGFs. Finally, we observed sites in IGF-II important for the ternary complex formation using various IGF-II mutants. These IGF-II mutants, which contained a substitution of Tyr27 for Leu, had extremely reduced activity. These results strongly suggest that: 1) the N domain, containing at least Glu52-Ala92, of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for binding to the acid-labile subunit both in the presence and absence of IGF-II; and 3) Tyr27 of IGF-II is important for the ternary complex formation. We have examined regions of rat IGF-binding protein-3 (IGFBP-3) important for complex formations using two kinds of deletion mutants, three kinds of chimera molecules between rat IGFBP-3 and rat IGFBP-2, and a synthetic peptide (41 residues, Glu52-Ala92) derived from rat IGFBP-3. Solid-phase binding assays using 96-well microtiter plates were designed to quantitate the relative binding affinities. It was found that not only the IGFBP-3 derivatives with the amino-terminal, cysteine-rich domain (N domain) but also the synthetic peptide maintained affinity for IGF-II. Ternary complex formation was observed with full-length IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domain (C domain) of which was derived from IGFBP-3, unlike the mutants lacking the C domain and the chimera IGFBPs, the C domain of which was derived from IGFBP-2. These results were confirmed by affinity cross-linking experiments. Furthermore, the IGFBP-3 derivatives that possessed the C domain of IGFBP-3 bound to the acid-labile subunit, even in the absence of IGFs. Finally, we observed sites in IGF-II important for the ternary complex formation using various IGF-II mutants. These IGF-II mutants, which contained a substitution of Tyr27 for Leu, had extremely reduced activity. These results strongly suggest that: 1) the N domain, containing at least Glu52-Ala92, of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for binding to the acid-labile subunit both in the presence and absence of IGF-II; and 3) Tyr27 of IGF-II is important for the ternary complex formation. Insulin-like growth factor (IGF) 1The abbreviations used are: IGF, insulin-like growth factor; rIGF, recombinant IGF; IGFBP, IGF-binding protein; ALS, acid-labile subunit; HRP, horseradish peroxidase; PhoA, phosphatase A; pAb, polyclonal antibody. -I and -II contain three disulfide bonds and have both amino acid sequence and tertiary structural homology with each other and with insulin (1Humbel R.E. Eur. J. Biochem. 1990; 190: 445-462Crossref PubMed Scopus (679) Google Scholar, 2Dodson E.J. Dodson G.G. Hodgkin D.C. Reynolds C.D. Can. J. Biochem. 1979; 57: 469-479Crossref PubMed Scopus (98) Google Scholar, 3Cooke R.M. Harvey T.S. Campbell I.D. Biochemistry. 1991; 30: 5484-5491Crossref PubMed Scopus (164) Google Scholar, 4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar). Unlike insulin, IGFs bind to a family of specific IGF-binding proteins (IGFBPs) that regulate IGF function in blood and body fluids (5Clemmons D.R. Growth Regul. 1992; 2: 80-87PubMed Google Scholar, 6Rechler M.M. Vitam. Horm. 1993; 47: 1-114Crossref PubMed Scopus (447) Google Scholar). There are six kinds of IGFBPs, designated IGFBP-1 to IGFBP-6, which have related primary structures (7Shimasaki S. Ling N. Prog. Growth Factor Res. 1991; 3: 243-266Abstract Full Text PDF PubMed Scopus (649) Google Scholar). Except for IGFBP-6, all human and rat IGFBPs contain 18 homologous cysteine residues in the amino-terminal and the carboxyl-terminal conserved domains (7Shimasaki S. Ling N. Prog. Growth Factor Res. 1991; 3: 243-266Abstract Full Text PDF PubMed Scopus (649) Google Scholar). Recently, it was reported that the mac25 gene encodes a preprotein (named IGFBP-7) of 277 amino acids, which contain the common IGFBP motif in the amino-terminal domain. In this domain, 11 of the usual 12 cysteines are conserved (8Swisshelm K. Ryan K. Tsuchiya K. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4472-4476Crossref PubMed Scopus (198) Google Scholar), and IGFBP-7 binds IGFs with specificity (9Oh Y. Nagalla S.R. Yamanaka Y. Kim H.-S. Wilson E. Rosenfeld R.G. J. Biol. Chem. 1996; 271: 30322-30325Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Although IGFBP-7 contains a total of 18 cysteines, consistent with the IGFBP-1 to IGFBP-5 molecules, the carboxyl-terminal domain contains only one homologous cysteine position. When IGFs are bound to IGFBP-3, an acid-labile subunit (ALS) can bind to the IGF/IGFBP-3 complex to form an IGF/IGFBP-3/ALS 150-kDa ternary complex (10Baxter R.C. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6898-6902Crossref PubMed Scopus (304) Google Scholar, 11Baxter R.C. Martin J.L. Beniac V.A. J. Biol. Chem. 1989; 264: 11843-11848Abstract Full Text PDF PubMed Google Scholar). A recent study also identified a binary complex of IGFBP-3/ALS in rat serum (12Lee C.Y. Rechler M.M. Endocrinology. 1995; 136: 668-678Crossref PubMed Google Scholar, 13Lee C.Y. Rechler M.M. Endocrinology. 1995; 136: 4982-4989Crossref PubMed Google Scholar). Through binding analyses using various IGFs mutants, it was reported that Glu6, Phe26, Phe48, Arg49, and Ser50 of human IGF-II were important for binding to IGFBPs (14Lüthi C. Roth B.V. Humbel R.E. Eur. J. Biochem. 1992; 205: 483-490Crossref PubMed Scopus (17) Google Scholar, 15Francis G.L. Aplin S.E. Milner S.J. McNeil K.A. Ballard F.J. Wallace J.C. Biochem. J. 1993; 293: 713-719Crossref PubMed Scopus (75) Google Scholar, 16Bach L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Abstract Full Text PDF PubMed Google Scholar). There is also information about binding regions of IGFBPs to IGFs derived from fragments of IGFBPs that exist in human and rat serum (6Rechler M.M. Vitam. Horm. 1993; 47: 1-114Crossref PubMed Scopus (447) Google Scholar). For example, amino-terminal 30-kDa fragments of IGFBP-3 obtained from human (17Zapf J. Kiefer M. Merryweather J. Musiarz F. Bauer D. Born W. Fischer J.A. Froesch E.R. J. Biol. Chem. 1990; 265: 14892-14898Abstract Full Text PDF PubMed Google Scholar) and rat (18Zapf J. Born W. Chang J.-Y. James P. Froesch E.R. Fischer J.A. Biochem. Biophys. Res. Commun. 1988; 156: 1187-1194Crossref PubMed Scopus (59) Google Scholar, 19Shimonaka M. Schroeder R. Shimasaki S. Ling N. Biochem. Biophys. Res. Commun. 1989; 165: 189-195Crossref PubMed Scopus (55) Google Scholar) serum were able to bind IGFs. Furthermore, an amino-terminal 15-kDa fragment derived from human plasma IGFBP-3 was detected by ligand blotting using125I-labeled IGF-I (20Sommer A. Maack C.A. Spratt S.K. Mascarenhas D. Tressel T.J. Rhodes E.T. Lee R. Roumas M. Tatsuno G.P. Flynn J.A. Gerber N. Taylor J. Cudny H. Nanney L. Hunt T.K. Spencer E.M. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: 715-728Google Scholar). These natural fragments corresponding to the amino-terminal domain of IGFBP-3 were isolated without disulfide reduction, suggesting that these domains are not linked by disulfide bonds (6Rechler M.M. Vitam. Horm. 1993; 47: 1-114Crossref PubMed Scopus (447) Google Scholar). In the case of the IGFs/IGFBP-3/ALS ternary complex formation, Tyr24 and the D-domain (residues 63–70) of human IGF-I are thought to be important (21Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Abstract Full Text PDF PubMed Google Scholar). However, the properties of IGFBP-3 and ALS for ternary complex formation are not understood well. We previously characterized IGFs binding to their receptors and biological activities of IGFs using various recombinant IGF-II mutants (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar, 23Hashimoto R. Fujiwara H. Higashihashi N. Enjoh-Kimura T. Terasawa H. Fujita-Yamaguchi Y. Inagaki F. Perdue J.F. Sakano K. J. Biol. Chem. 1995; 270: 18013-18018Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and confirmed the three-dimensional structure of recombinant IGF-II (4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar). In this study, we describe new solid-phase binding assays that can be performed easily using 96-well microtiter plates. Using these assays, we evaluated the binding properties of various mutants, chimeras, and a synthetic peptide derived from rat IGFBP-3 to analyze IGFs/IGFBP-3 and IGFs/IGFBP-3/ALS structure-function relationships. A rat pancreas cDNA (λgt 11) library of adult Sprague-Dawley females was purchased fromCLONTECH Laboratories, Inc. (Palo Alto, CA). The prokaryotic expression vector pTrc99A, pGEX-2T, DEAE-Sepharose CL-6B resin, SP-Sepharose F.F. resin, and NHS-activated HiTrap affinity column were from Pharmacia Biotech, Inc. (Uppsala, Sweden). Protein A affinity resin (PROSEP-A) was from Bioprocessing, Ltd. (Durham, United Kingdom) and DEAE-5PW 75 × 7.5 mm inside diameter was from Tosoh (Tokyo, Japan). A YMC-Pack Protein-RP column (250 × 4.6 mm inside diameter) was from YMC Co., Ltd. (Kyoto, Japan). A maleimide-activated horseradish peroxidase kit and disuccinimidyl suberate were purchased from Pierce. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) peroxidase substrate and microtiter plates (96-well) were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD) and Costar (Cambridge, MA), respectively. Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (anti-rabbit IgG-HRP), a biotinylation kit, and125I-labeled human recombinant IGF-II (2000 Ci/mmol) were obtained from Amersham Corp. All other chemicals were of the highest quality commercially available. Recombinant human IGF-II and IGF-II mutants of [Leu27]rIGF-II, [Leu43]rIGF-II, and [Arg54, Arg55]rIGF-II were expressed inEscherichia coli MC1061 using constructs and previously (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar). A of was by using synthetic were by an previously Y. Sato Y. Fujiwara H. Sakano K. Y. M. M. S. J. PubMed Scopus Google Scholar), and were confirmed by the F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) using a kit Japan). of IGFBP-3 and cDNA was performed by with to cDNA S. A. M. M. Ling N. Biochem. Biophys. Res. Commun. 1989; 165: PubMed Scopus Google Scholar, L. T. E.J. Rechler M.M. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar) using rat pancreas cDNA library (λgt 11) a the homology all rat IGFBPs, IGFBP-3 contained the three an amino-terminal and carboxyl-terminal domain in cysteine and an with IGFBP-3 mutants designated domain and N domain were by using the rat IGFBP-3 cDNA a A chimera and C domain were derived from IGFBP-3, IGFBP-2, and IGFBP-2, was In this chimera proteins designated and were by fragments from the and C domains using rat IGFBP-3 and rat cDNA All IGFBP-3 derivatives are in For the expression of IGFBP-3, domain, and in E. the phosphatase A sequence was and of pTrc99A, of these which were of their These expression were E. coli The were at in an with the were an and by at for The by L.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) from the was to with and to an IGF-II affinity resin column from an NHS-activated HiTrap affinity column to the IGFBP-3 was with acid and in and by Protein-RP × mm inside with a from to in The of only the IGFBP-3 N domain was expressed coli a with The which a was was using expression vector The was from a affinity resin using a to the The with mm reduced was and The N domain was from the by with in acid and The obtained N domain was previously Y. T. Enjoh T. Numata F. Sakano K. Biochem. 1992; PubMed Scopus Google Scholar) and using the IGF-II affinity column and of the six disulfide in the N domain of IGFBP-3 were by peptide that and amino-terminal amino acid sequence of isolated peptide fragments not to the identified disulfide bonds of and a peptide derived from was by an peptide using the formation J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). of rat serum were mm and a DEAE-Sepharose CL-6B column and an complex affinity column previously with R.C. Martin J.L. Beniac V.A. J. Biol. Chem. 1989; 264: 11843-11848Abstract Full Text PDF PubMed Google Scholar, R.C. J. Endocrinology. 1994; PubMed Scopus Google Scholar). For this study, IGF-II was to an NHS-activated HiTrap affinity and recombinant IGFBP-3 was used of IGFBP-3. Finally, the ALS was a DEAE-5PW column × 7.5 mm inside using a from to in mm containing ALS were and mm by The of each IGFBP-3, domain, N domain, the and ALS was confirmed by in the PubMed Scopus Google Scholar) by All of the were using an amino acid column to the acid of all proteins were also confirmed by using an was using a maleimide-activated kit to the IGF-II was to a the primary and was with maleimide-activated ALS was primary using a biotinylation kit to the of IGFBP-3 the peptide were to at polyclonal and pAb, was from the using a affinity column to the and mm All solid-phase binding assays were with 96-well microtiter plates using binding in For all assays, the plates were with proteins in and with the containing serum All were with the containing proteins were in the containing serum and all were performed at For the binding of IGFBP-3 were at The were and for at The were and of a of and of each were to each well. The plates were for and and of acid) substrate & Perry were the of the at was using CA). study complex formation with ALS, using ALS were were with of and sites were of ALS were and for to ALS the relative of the IGFBP-3 derivatives to form ternary the solid-phase binding of ALS were of ALS, of a of and of various of IGFBP-3 derivatives were for to form the ternary The of the complex in the was the binding of IGFBP-3 derivatives to ALS in the absence of IGFs the IGFBP-3/ALS solid-phase binding various of IGFBP-3 derivatives were with the the the of the complex was detected by with of for by and of a of anti-rabbit for the of the IGF-II mutants to form the ternary the IGFs/IGFBP-3/ALS solid-phase binding of ALS were to of the IGFBP-3/ALS complex in the absence of IGFs. of ALS, of various of IGF-II mutants and of IGFBP-3 were for The ternary were detected using the and anti-rabbit IGFBP-3 the peptide was at in of mm containing and IGF-II in the absence the binary in the presence the ternary of of an of suberate were and the was for The cross-linking was by of were by acid at a of and to a by IGFBP-3, two kinds of deletion mutants, three kinds of chimera IGFBPs, the and rat serum ALS were and their was confirmed by not and by The chimera IGFBPs were expressed using the expression vector the IGFBP-3, but all chimera IGFBPs The were also detected by blotting using and by ligand blotting using not the results of amino-terminal amino acid sequence it was found that all chimera IGFBPs contained the amino-terminal sequence of the primary sequence and an The sequence is to of the in the Recombinant rat IGFBP-3 expressed coli not the it is that IGFBP-3 have contained the sequence at Using the solid-phase binding the relative of IGFBP-3 mutants and the peptide to IGF-II were from their and with that of IGFBP-3, which was to The chimera IGFBPs of the relative but the isolated domain N domain A in affinity was observed between domain and N domain. In the case of the it only affinity relative to IGFBP-3, an binding to IGFBP-3. the IGFBP-3 mutants and the peptide binding to affinity cross-linking was performed using IGF-II. The IGFBP-3, domain, N domain, and the peptide all consistent with cross-linking IGF-II The using only ALS not form a complex These results are consistent with the that the N domain of IGFBP-3, and at least a of the peptide is important for binding to binding of IGFBP-3 derivatives and peptide for were from the binding in of were from the binding in in a new of the binding of IGFBP-3 deletion mutants to IGF-II by affinity by cross-linking of IGF-II and domain N domain and peptide a of cross-linking of IGF-II and ALS Using the solid-phase binding activities for the ternary complex formation of IGFBP-3 mutants and the peptide be In this of ALS was of ALS ALS not a using of IGFBP-3 not IGFBP-3 ternary complex formation between and the the binding in However, the domain, N domain, the which all had affinity for were observed to form ternary the chimeras, only ternary complex formation The activities of the chimera IGFBPs for the ternary complex formation were also examined by affinity cross-linking using125I-labeled IGF-II and ALS The derived from the binary complex with IGF-II were detected with IGFBP-3 and all chimera IGFBPs, derived from the ternary complex were detected only with IGFBP-3 and which contains only the C domain from IGFBP-2, not this that the domain is not for ternary complex of chimera IGFBPs activities in ternary complex formation by affinity by cross-linking IGF-II and IGFBP-3 and and and with and without and ALS derived from the ternary complex the binary complex and IGF-II are with the IGFBP-3/ALS binary the was to the solid-phase binding of IGFBP-3 were to the and the IGFBP-3/ALS binary complex was detected by by anti-rabbit confirmed that the bind to all chimera IGFBPs by not in IGFBP-3 and bound to ALS in a The other chimera IGFBPs and had binding to These results strongly that the C domain of IGFBP-3 an important for the binding of IGFBP-3 to ALS in the presence and absence of IGFs. The relative of IGF-II mutants to IGFBP-3 were by the solid-phase binding from their in A and with that of which was to The of [Leu27]rIGF-II, [Leu43]rIGF-II, [Arg54, and for IGFBP-3 were and relative to IGF-II. The from the three mutants are to reported and L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Abstract Full Text PDF PubMed Google Scholar). The to form ternary with these IGF-II mutants were examined with the IGFs/IGFBP-3/ALS solid-phase binding The and [Arg54, Arg55]rIGF-II the activities relative to IGF-II. However, the two mutants with substitution of Tyr27 to ternary complex formation, suggesting that Tyr27 of IGF-II to the binding in the ternary of the recombinant IGFBP-3 and the the expression with sequence was used with the of the N domain All of the expressed proteins were by IGF-II affinity column a binding that and of relative affinities. The IGFBP-3 derivatives used in this study were for chimera chimera IGFBP contained a form and a with two amino from the amino Although the is it is that the of each chimera IGFBP was an the sequence is the other IGFBP-3 and the domain were also expressed with the and only We the that IGFBP-3 and the domain also be at the at the solid-phase binding and affinity cross-linking the deletion mutants of IGFBP-3, the domain, and the N domain and for relative to full-length IGFBP-3. that a 30-kDa IGFBP-3 in plasma affinity for IGF-I plasma IGFBP-3 M. P. C. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: Scholar), and a carboxyl-terminal IGFBP-3 from rat serum also affinity for IGF-I C. J. Froesch E.R. J. Biochem. Biophys. Res. Commun. 1991; PubMed Scopus Google Scholar). The IGFBP-3 from by IGFBP-3 and reduced affinity for of the and the of the IGFs M. P. C. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: Scholar). obtained from the binding of IGFBP-3 deletion mutants to IGF-II are in with that of and form of IGFBP-3. Furthermore, we found that the peptide Glu52-Ala92) bind to IGF-II. In a study, the amino-terminal amino of human IGFBP-3 affinity for IGF-I (20Sommer A. Maack C.A. Spratt S.K. Mascarenhas D. Tressel T.J. Rhodes E.T. Lee R. Roumas M. Tatsuno G.P. Flynn J.A. Gerber N. Taylor J. Cudny H. Nanney L. Hunt T.K. Spencer E.M. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: 715-728Google Scholar). The peptide is the fragment of the IGFBP-3 derivatives found to bind to IGFs. peptide was designed to two which were identified by peptide The bonds in the N domain not be of the cysteine-rich at we one peptide that contained the Although the peptide of the affinity for IGF-II relative to the N domain, the peptide disulfide bonds were reduced and not binding affinity not the structure maintained by the bonds are thought to be important for the binding However, to the relative of and the domain for the presence of only the N domain is important but not for For example, the and C domains important for of the N domain. For ternary complex formation, it was found that only the chimera IGFBP, which possessed the C domain derived from IGFBP-3 ternary complex formation in the solid-phase binding the mutants lacking the C the domain, the N domain, and the were not in the affinity cross-linking and it was found that the derived from the ternary complex were observed using only IGFBP-3 and Although IGF-II and ALS were not observed derived from the binary complex IGF-II and ALS were detected in the presence of IGFBP-3 in A study also that a complex was detected by affinity cross-linking in the presence of IGFBP-3 (10Baxter R.C. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6898-6902Crossref PubMed Scopus (304) Google Scholar). It is that IGF-II not have a binding affinity for ALS but be ALS in the ternary complex that the can IGF-II and ALS in the presence of IGFBP-3. the that IGFBP-3 and can form the ternary complex but that the domain and it is that the C domain of IGFBP-3 is for binding to The results of the IGFBP-3/ALS solid-phase binding also suggest that the C domain of IGFBP-3 is important for binding to ALS, even in the absence of IGF-II. In this study, we not an affinity cross-linking to chimera IGFBPs binding to the binding of IGFBP-3 to ALS, only the C domain expressed in E. be used in the solid-phase binding and affinity cross-linking experiments. we confirmed the three-dimensional structure of recombinant IGF-II (4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar) and identified the binding sites of IGF-II for their receptors (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar, 23Hashimoto R. Fujiwara H. Higashihashi N. Enjoh-Kimura T. Terasawa H. Fujita-Yamaguchi Y. Inagaki F. Perdue J.F. Sakano K. J. Biol. Chem. 1995; 270: 18013-18018Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and for IGFBPs L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Abstract Full Text PDF PubMed Google Scholar) using various IGF-II mutants. We also observed IGF-II sites important for the ternary complex formation using IGF-II mutants. that IGF-II is important for binding to insulin and IGF-I receptors (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar) and not for binding to the and that the Tyr27 is at the from a binding for IGFBPs, which Glu6, Phe48, Arg49, and Ser50 (4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar). In this study, the results of the IGFs/IGFBP-3/ALS solid-phase binding suggest that of Tyr27 to not ternary complex formation. results have obtained by other using IGF-I mutants in which Tyr24 with reduced the ternary complex formation (21Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Abstract Full Text PDF PubMed Google Scholar). Tyr24 of IGF-I is also important for binding to insulin and IGF-I receptors M.A. Bayne M.L. D. and of Insulin-like Growth and Publishing Google Scholar). the Tyr27 of which is important for binding to insulin and IGF-I only with ALS and is a to the Glu6, Phe48, Arg49, and Ser50 residues, which bind the amino-terminal domain of IGFBP-3, the carboxyl-terminal domain of IGFBP-3 binds to ALS, even in the absence of IGF-II. We M. J. F. and Y. Fujita-Yamaguchi for and We also Y. M. and K. Sato for