A Novel Binding Site for the Human Insulin-like Growth Factor-II (IGF-II)/Mannose 6-Phosphate Receptor on IGF-II

Abstract

The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe19 and Leu53 are critical for IGF2R binding, as are residues Thr16 and Asp52. Furthermore, Thr16 was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R. The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe19 and Leu53 are critical for IGF2R binding, as are residues Thr16 and Asp52. Furthermore, Thr16 was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R. The mammalian insulin-like growth factor-II (IGF-II) 5The abbreviations used are: IGF, insulin-like growth factor; IGF2R, IGF-II/cation-independent mannose 6-phosphate receptor; IGF-1R, type 1 IGF receptor; HPLC, high pressure liquid chromatography. 5The abbreviations used are: IGF, insulin-like growth factor; IGF2R, IGF-II/cation-independent mannose 6-phosphate receptor; IGF-1R, type 1 IGF receptor; HPLC, high pressure liquid chromatography./cation-independent mannose 6-phosphate receptor (IGF2R) has multiple functions due to its ability to interact with a wide variety of ligands. This transmembrane receptor has 15 homologous extracellular repeat domains and a short intracellular domain, which lacks kinase activity (1Scott C.D. Firth S.M. Horm. Metab. Res. 2004; 36: 261-271Crossref PubMed Scopus (44) Google Scholar). The IGF2R plays a major role in targeting mannose 6-phosphate-labeled proteins via the trans-Golgi network and from the cellular membrane to lysosomes for degradation (2Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (778) Google Scholar). It also binds and internalizes IGF-II, thereby maintaining correct levels of IGF-II locally and in the circulation. These functions are central in the processes of embryonic development and normal growth. Mice lacking the IGF2R have increased levels of circulating IGF-II, are born larger, and die soon after birth due to cardiac hyperplasia. This phenotype is overcome by concomitant deletion of the IGF-II gene (3Ludwig T. Eggenschwiler J. Fisher P. D'Ercole A.J. Davenport M.L. Efstratiadis A. Dev. Biol. 1996; 177: 517-535Crossref PubMed Scopus (398) Google Scholar). Likewise, loss of IGF2R activity in many cancers has been associated with increased tumor cell growth and tumor progression. This can arise from the loss of heterozygosity or mutation of the receptor (4De Souza A.T. Hankins G.R. Washington M.K. Orton T.C. Jirtle R.L. Nat. Genet. 1995; 11: 447-449Crossref PubMed Scopus (331) Google Scholar, 5Devi G.R. De Souza A.T. Byrd J.C. Jirtle R.L. MacDonald R.G. Cancer Res. 1999; 59: 4314-4319PubMed Google Scholar), leading to an increase in bioavailable IGF-II, which then acts via the type 1 IGF receptor (IGF-1R) to promote cancer growth and survival. The extracellular domains of the IGF2R share significant similarity with the single extracellular domain of the cation-dependent mannose 6-phosphate receptor (2Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (778) Google Scholar). Domains 3 and 9 and more recently domain 5 of the IGF2R were shown to contain mannose 6-phosphate binding sites (6Hancock M.K. Haskins D.J. Sun G. Dahms N.M. J. Biol. Chem. 2002; 277: 11255-11264Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Hancock M.K. Yammani R.D. Dahms N.M. J. Biol. Chem. 2002; 277: 47205-47212Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Reddy S.T. Chai W. Childs R.A. Page J.D. Feizi T. Dahms N.M. J. Biol. Chem. 2004; 279: 38658-38667Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and act as binding sites for ligands, including the urokinase plasminogen activator receptor, plasminogen, transforming growth factor-β, and retinoic acid. A distinct IGF-II binding site is located on domain 11, and the additional presence of domain 13 is required for high affinity IGF-II binding equivalent to intact IGF2R (9Dahms N.M. Wick D.A. Brzycki-Wessell M.A. J. Biol. Chem. 1994; 269: 3802-3809Abstract Full Text PDF PubMed Google Scholar, 10Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, 11Schmidt B. Kiecke-Siemsen C. Waheed A. Braulke T. von Figura K. J. Biol. Chem. 1995; 270: 14975-14982Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The crystal structure of the IGF2R domain 11 has been solved (12Brown J. Esnouf R.M. Jones M.A. Linnell J. Harlos K. Hassan A.B. Jones E.Y. EMBO J. 2002; 21: 1054-1062Crossref PubMed Scopus (89) Google Scholar), and specific mapping of the IGF-II binding site on this domain has recently been reported (13Zaccheo O.J. Prince S.N. Miller D.M. Williams C. Kemp C.F. Brown J. Jones E.Y. Catto L.E. Crump M.P. Hassan A.B. J. Mol. Biol. 2006; 359: 403-421Crossref PubMed Scopus (26) Google Scholar). Residues corresponding to the mannose 6-phosphate binding site of the cation-dependent mannose 6-phosphate receptor were mutated to Ala, and Tyr1542, Thr1570, Phe1567, and Ile1572 within this hydrophobic patch were shown to be critical for IGF-II binding. Ile1572 had previously been identified as essential for IGF-II binding (10Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar). Interestingly, mutation of Glu1544 (which lies at the edge of this binding site) to Ala or Lys leads to an increase in binding affinity (13Zaccheo O.J. Prince S.N. Miller D.M. Williams C. Kemp C.F. Brown J. Jones E.Y. Catto L.E. Crump M.P. Hassan A.B. J. Mol. Biol. 2006; 359: 403-421Crossref PubMed Scopus (26) Google Scholar). Although the binding site on domain 11 has been mapped, it is not yet clear whether IGF-II contacts domain 13. IGF-I and IGF-II share >60% sequence identity, yet the IGF2R only binds IGF-II with high affinity. Little is known about this interaction, since relatively few IGF-II mutants have been made, and a structure of the IGF2R·IGF-II complex has not been solved. Residues Phe48, Arg49, and Ser50 have been strongly implicated in IGF2R binding (14Sakano 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). However, these residues are conserved between IGF-I and IGF-II and therefore would not drive the specificity of binding. Ala54 and Leu55 of IGF-II differ from the corresponding residues in IGF-I (Arg55, Arg56) and are important for IGF2R binding (15Forbes B.E. McNeil K.A. Scott C.D. Surinya K.H. Cosgrove L.J. Wallace J.C. Growth Factors. 2001; 19: 163-173Crossref PubMed Scopus (12) Google Scholar). In addition, we recently showed that IGF-II residues in the nonconserved C domain are important for IGF2R binding and play a role in conferring the specificity of binding (16Roche P. Brown J. Denley A. Forbes B.E. Wallace J.C. Jones E.Y. Esnouf R.M. Proteins. 2006; 64: 758-768Crossref PubMed Scopus (11) Google Scholar). However, the effects of mutation on residue Ala54 or Leu55 or the C domain are insufficient to account for the complete lack of binding by IGF-I, suggesting that another determinant provides the IGF-II specificity. In this study, we have significantly added to our understanding of the IGF2R binding site on IGF-II using a mutagenesis approach and surface plasmon resonance with isolated IGF2R domains. We have revealed a novel hydrophobic patch critical for IGF2R binding, which encompasses residues Phe19 and Leu53 and includes Thr16 and Asp52. We have also identified the major determinant on IGF-II responsible for the specificity of IGF2R binding. Materials—Oligonucleotides (Table 1) were purchased from Geneworks Pty. Ltd. (Adelaide, South Australia). Restriction enzymes were from New England Biolabs (Hitchin, UK) or Geneworks Pty. Ltd. Long™R3IGF-I, human IGF-I, and IGF-II were purchased from GroPep Ltd. (Adelaide, South Australia). Greiner Lumitrac 600 96-well plates were from Omega Scientific (Tarzana, CA). Human insulin was purchased from Novo Nordisk (Bagsvaerd, Denmark). The DELFIA europium-labeling kit and DELFIA enhancement solution were purchased from PerkinElmer Life Sciences. Europium-labeled IGF-II was produced as described by Denley et al. (17Denley A. Bonython E.R. Booker G.W. Cosgrove L.J. Forbes B.E. Ward C.W. Wallace J.C. Mol. Endocrinol. 2004; 18: 2502-2512Crossref PubMed Scopus (162) Google Scholar) according to the manufacturer's instructions. P6 cells (BALB/c3T3 cells overexpressing the human IGF-1R) (18Pietrzkowski Z. Lammers R. Carpenter G. Soderquist A.M. Limardo M. Phillips P.D. Ullrich A. Baserga R. Cell Growth Differ. 1992; 3: 199-205PubMed Google Scholar) were a kind gift from Professor R. Baserga (Philadelphia, PA).TABLE 1Oligonucleotides used to generate mutant IGF-II cDNA constructsMutationSequenceE12K5′-CTG TGC GGT GGT AAA CTG GGT CTG CTG CTG CTG CTG TGC GGT CTG TGC GGT CTG TGC GGT CTG CTG TGC GGT CTG CTG TGC GGT TGC TGC TGC TGC TGC CTG CTG in a of Human IGF-II IGF was by et al. R. P. M. J. Wallace J.C. M. J. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar), and the cDNA IGF-II was the as previously described McNeil K.A. Wallace J.C. J. PubMed Scopus Google Scholar). The mutagenesis kit was used to the IGF-II (Table were for IGF-II mutants were as proteins with the 11 of growth after were isolated as previously described R. P. M. J. Wallace J.C. M. J. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar). and of IGF-II the IGF-II were as described McNeil K.A. Wallace J.C. J. PubMed Scopus Google Scholar) with from a were in and at were on a in using the but with the IGF-II were identified by and and to in 5 1 and to of the and a were as previously described S. Wallace J.C. 2001; PubMed Scopus Google Scholar, B. M.A. K. Wallace J.C. J. Mol. Endocrinol. 1996; PubMed Scopus Google Scholar). proteins were by and and and were shown to have the correct and to be of analogues was by with of Wallace J.C. J. 1995; PubMed Scopus Google Scholar). were on a were from to with a using a and were for were in a with a were by of the of In to of the from in of were to the at 2003; PubMed Scopus Google Scholar). binding were using an to that described for growth factor binding to the growth factor receptor M. A. A. B. F. P. A. W. Y. 2002; PubMed Scopus (26) Google Scholar) and in A. Bonython E.R. Booker G.W. Cosgrove L.J. Forbes B.E. Ward C.W. Wallace J.C. Mol. Endocrinol. 2004; 18: 2502-2512Crossref PubMed Scopus (162) Google P6 cells were to with 1 and 1 for 1 at were for at at and then were added to of a Greiner Lumitrac 600 previously with M.A. Lammers R. Ullrich A. B. R.A. T. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). of IGF-II were added to with of and for at were with and and with and then DELFIA enhancement solution was was using and with a Australia). IGF2R domain 11 and IGF2R domains were as described previously (12Brown J. Esnouf R.M. Jones M.A. Linnell J. Harlos K. Hassan A.B. Jones E.Y. EMBO J. 2002; 21: 1054-1062Crossref PubMed Scopus (89) Google Scholar, P. Brown J. Denley A. Forbes B.E. Wallace J.C. Jones E.Y. Esnouf R.M. Proteins. 2006; 64: 758-768Crossref PubMed Scopus (11) Google Scholar). was as in and was produced as in a mammalian The approach was also used for of IGF2R domains 11 and IGF2R domains and IGF2R domains The required of were by using which also a the R.L. Harlos K. Williams J. Mol. Biol. PubMed Scopus Google Scholar), the was by and cells P. Mol. Cell Biol. PubMed Scopus Google Scholar) using proteins were by affinity and and of IGF2R Using IGF2R was to the using a to that previously described (16Roche P. Brown J. Denley A. Forbes B.E. Wallace J.C. Jones E.Y. Esnouf R.M. Proteins. 2006; 64: 758-768Crossref PubMed Scopus (11) Google Scholar). the IGF2R to be was in at and over the surface in The were then by 1 A cell was used as a on were at using a within the South Australia). The and were to the surface to a resonance of and were to of and The of surface was for for the which was IGF-II, IGF-I, and IGF analogues were over the at the IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, and IGF-II at of and IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, and IGF-II, IGF-II, IGF-I, and IGF-I at and in for at a of to of in was at the for 15 cells were by of of cell were from to account for due to the were using the and were to a affinity The the affinity of the interaction at The and were using a and were from of the This a binding interaction with a binding, in and This in the of the and has been previously used for of binding to IGF2R (13Zaccheo O.J. Prince S.N. Miller D.M. Williams C. Kemp C.F. Brown J. Jones E.Y. Catto L.E. Crump M.P. Hassan A.B. J. Mol. Biol. 2006; 359: 403-421Crossref PubMed Scopus (26) Google Scholar). mutant was in in a of The affinity from on for IGF-II on was and a between was on with mutants and of IGF-II novel IGF-II analogues were to the IGF2R binding had previously been and were to the of with Ala IGF-II, IGF-II, IGF-II, and The role of at was by with and The of at was by with was to the equivalent residue in insulin and the hydrophobic residue of was used in of at was with The equivalent mutation in IGF-I identified as the mutation in the IGF-I leads to an IGF-I with binding M. A.M. Y. J. S. Denley A. Forbes B. J. Endocrinol. Metab. PubMed Scopus Google Scholar). The of the was by with and was used to the of the of can be identified by a significant in the R. C. B. B. J. A. De P. S. 1999; PubMed Scopus Google Scholar). The analogues from IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, IGF-II, and IGF-II in for IGF-II, IGF-II, and IGF-II were but the to have little on and we that correct was of was by binding. IGF-I has a affinity for the R. C. B. B. J. A. De P. S. 1999; PubMed Scopus Google Scholar), and a in affinity is for IGF-II Y. R.G. J. 1991; PubMed Scopus Google Scholar). C. K. A. J. C. and B. of the mutants that had that from IGF-II and the ability to the with high affinity IGF-II had the in yet its binding affinity was only IGF-II (Table We therefore this mutant is but the in of IGF-II binding to the by IGF-II analogues The to that of IGF-II binding to the IGF-II is and are the and from to in a Although the for IGF-II, IGF-II, and IGF-II, binding was significantly This was not since residues to IGF-I residues in the binding surface that is essential for binding P. J. Nat. Rev. 2002; PubMed Scopus Google Scholar). of in IGF-I to in to also in a in binding affinity J. 1995; PubMed Scopus Google Scholar). of IGF-I or insulin in to or receptor binding K. B. S. M.A. PubMed Scopus Google Scholar, A. McNeil K.A. H. Wallace J.C. M. Forbes B.E. Mol. Endocrinol. 19: PubMed Scopus Google Scholar). IGF2R that of the analogues were we then used surface plasmon resonance to ability to IGF2R The IGF2R affinity and and high affinity and of and for IGF-II binding to and were to (13Zaccheo O.J. Prince S.N. Miller D.M. Williams C. Kemp C.F. Brown J. Jones E.Y. Catto L.E. Crump M.P. Hassan A.B. J. Mol. Biol. 2006; 359: 403-421Crossref PubMed Scopus (26) Google Scholar, P. Brown J. Denley A. Forbes B.E. Wallace J.C. Jones E.Y. Esnouf R.M. Proteins. 2006; 64: 758-768Crossref PubMed Scopus (11) Google Scholar) (Table The for IGF-II binding to the and were and (Table These were also to (16Roche P. Brown J. Denley A. Forbes B.E. Wallace J.C. Jones E.Y. Esnouf R.M. Proteins. 2006; 64: 758-768Crossref PubMed Scopus (11) Google Scholar, J. G. Hassan A.B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google of IGF-II analogues binding to the affinity IGF2R were using a affinity as and are of IGF-II analogues to IGF-II are also of IGF-II of binding at were using the as were using the as were using the as in a of IGF-II analogues binding to the high affinity IGF2R were using a as and are of IGF-II analogues to IGF-II are also of IGF-II of binding at in a mutants were for ability to the IGF2R mutant to affinity and with a affinity and 3 and was little on IGF2R binding with the and IGF-II IGF-II and IGF-II had and for the but had little on of Phe19 and IGF-II analogues binding to affinity IGF2R were with and and and IGF-II and IGF-II at and IGF-II, IGF-II, IGF-II, IGF-II, and IGF-II at were over as described in the to In contrast, binding was with IGF-II, IGF-II, or IGF-II at a of However, IGF-II had and IGF-II for and This would that Phe19 is involved in a hydrophobic interaction with the IGF2R, and the with a can binding. Leu53 and Thr16 are also critical for binding to the affinity with binding at a of In addition, mutation of to or has a significant on binding to and with the by with affinity with and mutant to high affinity and to a and and was little on IGF2R binding by the and with more for and Interestingly, IGF-II to and with only affinity IGF-II, was a of the IGF-II mutation on binding to the high affinity binding was with IGF-II, IGF-II, IGF-II, IGF-II, and IGF-II to and IGF-II also showed binding over IGF-II for the high affinity due to However, this was only on the high affinity to affinity with the affinity The and IGF-II had a in binding to the high affinity IGF2R as was with and with the the additional IGF-I mutant was using a approach as previously described B. M.A. K. Wallace J.C. J. Mol. Endocrinol. 1996; PubMed Scopus Google Scholar). significant were by of at this and binding affinity was to IGF-I not suggesting that IGF-I was Interestingly, is binding of IGF-I to IGF2R using surface plasmon However, the IGF-I with a affinity IGF-II A was for the high affinity and and the affinity and not the lack of a structure of the complex and only IGF-II mutagenesis we a study at significantly our understanding of the interaction and the the specificity of the A series of novel IGF-II analogues were produced and for binding to IGF2R The IGF2R domain 11, which is essential for IGF-II binding, or domains 11 and which are required to high affinity binding (9Dahms N.M. Wick D.A. Brzycki-Wessell M.A. J. Biol. Chem. 1994; 269: 3802-3809Abstract Full Text PDF PubMed Google Scholar, 10Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, 11Schmidt B. Kiecke-Siemsen C. Waheed A. Braulke T. von Figura K. J. Biol. Chem. 1995; 270: 14975-14982Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The binding a IGF-II surface important for interaction with the IGF2R. This surface encompasses residues and Leu53 of IGF-II, which a patch This binding surface lies to residues Ala54 and previously identified as important for IGF2R binding (15Forbes B.E. McNeil K.A. Scott C.D. Surinya K.H. Cosgrove L.J. Wallace J.C. Growth Factors. 2001; 19: 163-173Crossref PubMed Scopus (12) Google Scholar), but is from residues Phe48, Arg49, and also reported to be involved in IGF2R binding (14Sakano 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). The in IGF2R binding of the hydrophobic residues Phe19 and with the is with the that IGF-II binds to the IGF2R using hydrophobic (10Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, O.J. Prince S.N. Miller D.M. Williams C. Kemp C.F. Brown J. Jones E.Y. Catto L.E. Crump M.P. Hassan A.B. J. Mol. Biol. 2006; 359: 403-421Crossref PubMed Scopus (26) Google Scholar). of Phe19 with including the is to IGF2R binding. However, an increase in binding affinity to and high affinity IGF2R is by with a residue with a hydrophobic patch on the IGF2R is critical for IGF-II binding (10Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, O.J. Prince S.N. Miller D.M. Williams C. Kemp C.F. Brown J. Jones E.Y. Catto L.E. Crump M.P. Hassan A.B. J. Mol. Biol. 2006; 359: 403-421Crossref PubMed Scopus (26) Google Scholar). the of by with or that is with the with the Lys also is important at this for IGF2R binding. A significant in binding was for of residues that important for binding and had little or on IGF2R binding, and it is that are located on the of the IGF-II This is with that mutation of and also had little or on IGF2R binding, a significant in binding (14Sakano 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, D.M. C. Res. 1991; PubMed Scopus Google Scholar). Interestingly, of the that had an on IGF2R binding binding to the affinity and high affinity IGF2R We can therefore that these residues are with domain 11 and our the that the role of domain 13 is to the in high affinity binding (9Dahms N.M. Wick D.A. Brzycki-Wessell M.A. J. Biol. Chem. 1994; 269: 3802-3809Abstract Full Text PDF PubMed Google Scholar, 10Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, 11Schmidt B. Kiecke-Siemsen C. Waheed A. Braulke T. von Figura K. J. Biol. Chem. 1995; 270: 14975-14982Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Thr16 of IGF-II is the only residue in this study that from its equivalent residue in IGF-I and therefore can be used to the IGF-II binding of the IGF2R. have identified that and the C domain of IGF-II to the specificity of the However, mutation of Ala54 or Leu55 to the equivalent residues in IGF-I and only in a binding affinity (15Forbes B.E. McNeil K.A. Scott C.D. Surinya K.H. Cosgrove L.J. Wallace J.C. Growth Factors. 2001; 19: 163-173Crossref PubMed Scopus (12) Google Scholar). the IGF-II C domain with the IGF-I C domain only binding by (16Roche P. Brown J. Denley A. Forbes B.E. Wallace J.C. Jones E.Y. Esnouf R.M. Proteins. 2006; 64: 758-768Crossref PubMed Scopus (11) Google Scholar). and the IGF-II C domain contain determinants important for IGF2R binding and play a role in are not the major mutation of Thr16 to Ala in a loss in binding to IGF2R only a in we this residue the major determinant for the specificity of the In of we showed that IGF-I to the IGF2R with affinity IGF-II is binding of IGF-I on or high affinity IGF2R The ability of the IGF2R to IGF-II in after the of and with the of development A.J. J.F. Z. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The mannose 6-phosphate of including and are to IGF-II due to in the sequence of the IGF-II binding site Byrd J.C. Jirtle M.K. MacDonald R.G. Jirtle R.L. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). Thr16 is conserved in IGF-II of mammalian in which the IGF2R has an IGF-II binding Thr16 play a role in IGF2R binding. Thr16 of IGF-II is in a and is this not in IGF-I, since the corresponding residue is more and of a surface The presence of the and at also be the structure in this are significant between IGF-I and IGF-II in the the residues and residues identified in this study to be important for IGF2R binding These from the of the critical residue in In we have a novel binding surface on IGF-II that is important for binding to domain 11 of the IGF2R and that high affinity binding is hydrophobic We have also identified Thr16 as the major determinant of IGF-II binding specificity for the IGF2R. This study provides understanding of the interaction, which is responsible for the of IGF-II in normal development and plays a significant role in tumor