Keeping IGF-II under control: Lessonsfrom the IGF-II–IGF2R crystal structureJames Brown1, E. Yvonne Jones1 and Briony E. Forbes2

1 Cancer Research UK Receptor Structure Research Group, Division of Structural Biology, Wellcome Trust Centre for Human

Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK2 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, 5005

Review

Glossary

Cation dependent mannose-6-phosphate receptor (CD-MPR): a �45 kDa

protein containing a single transmembrane domain. Together with the cation

independent-MPR (also known as IGF2R), it is a member of the p-type lectin

family.

Imprinting: this process results in differential expression of a gene depending

on whether it was maternally or paternally inherited. When a gene is paternally

imprinted, an allele is inherited from the father but is not expressed in

offspring. Similarly, when a gene is maternally imprinted, an allele is inherited

from the mother but is not expressed in offspring. Mouse IGF2R is expressed

from the maternal allele whereas IGF2 is expressed from the paternal allele.

Insulin-like growth factors (IGFs): small polypeptides related in sequence and

structure to insulin. IGF-I and IGF-II share over 75% sequence identity. Both are

important for foetal growth and development; IGF-I also contributes signifi-

cantly to growth hormone controlled postnatal growth.

Loss of heterozygosity (LOH): loss of function of one allele when the other

allele has been previously inactivated. LOH of IGF2R arises in cancers, e.g.

colon cancer, and is a result of somatic mutation in the maternal allele (IGF2R

is paternally imprinted).

Lysosomes: organelles that contain digestive enzymes (acid hydrolases) in an

Insulin-like growth factor-II (IGF-II) is a key regulator ofcell growth, survival, migration and differentiation. Itspivotal role in these processes requires tight regulationof both expression and activity. The type 1 IGF receptortyrosine kinase (IGF-1R) mediates IGF-II actions, and afamily of six high affinity IGF binding proteins (IGFBPs)regulates IGF-II circulating half-life and its availability tobind IGF-1R. In addition, the type 2 IGF receptor (IGF2R;also called the cation-independent mannose-6-phos-phate receptor) modulates the circulating and tissuelevels of IGF-II by targeting it to lysosomes for degra-dation. The recently elucidated crystal structure of IGF-II–IGF2R complex provides new insight into IGF-II regu-lation, and reveals a common binding surface on IGF-IIfor the regulatory proteins, IGF2R and the IGFBPs.

Biological functions of IGF-II, the IGF2R and IGFBPsInsulin-like growth factor-II (IGF-II; Box 1) promotes cellgrowth, survival, migration and differentiation via thetype 1 IGF tyrosine kinase receptor (IGF-1R) [1]. It alsostimulates mitogenic responses via the insulin receptorisoform-A (IR-A). IR-A arises from alternative splicing ofinsulin receptor (IR) and lacks the 12 amino acids encodedby exon 11 [2]. This isoform has high affinity for IGF-II aswell as insulin, and is predominantly expressed in thefoetus and by cancer cells [2].

IGF-II is important for foetal growth and development.In mice, Igf2 knockout leads to a significant reduction inbody size (�60% of normal) [3], and placental specificknockout demonstrated a critical role in that tissue’s de-velopment and function [4]. IGF2 is an imprinted gene. Inhumans, disruption of paternal allele expression results inSilver-Russell syndrome, which is characterized by foetalgrowth retardation and short stature [5,6]. By contrast,IGF-II overexpression due to a polymorphism that dis-rupts imprinting leads to overgrowth and Beckwith-Wie-deman syndrome [3,7]. Loss of IGF2 imprinting alsopromotes proliferation and survival in postnatal diseasesincluding Wilm’s tumour and colon cancer [8]. In rodents,circulating IGF-II levels are greatly reduced after birth,thuspointing to its importance in the foetus. Interestingly,however, IGF-II serum levels remain at high concen-trations in adult humans and the significance of thisobservation is not yet understood. IGF-II is required forhuman embryonic stem cell survival and self-renewal [9],and is also important in the adult for differentiation

Corresponding author: Forbes, B.E. (briony.forbes@adelaide.edu.au).

612 0968-0004/$ – see front matter . Crown Copyright � 2009 Published by Elsevier Lt

processes in muscle [10], brain [11] and brown adiposetissue [12].

IGF2R binds a large number of ligands including lyso-somal enzymes, IGF-II, transforming growth factor-b(TGF-b), granzyme B, uPAR and plasminogen, glycosy-lated LIF and retinoids. Intracellular IGF2R (�90% ofthe total produced) facilitates the trafficking of lysosomalenzymes between the trans-Golgi network, endosomes andlysosomes [13]. By contrast, IGF2R localized on the cellsurface regulates circulating and local levels of IGF-II,thereby influencing the actions of IGF-II, and acting asa growth inhibitor [13–16]. Cell surface-localized IGF2Ralso regulates IGF-II-mediated migration; such a role isespecially noticeable for human extravillous trophoblasts[17] and in cancer cell migration and invasion [15,18,19]. Asoluble form of IGF2R arises after cleavage from the cellsurface [20]. Soluble IGF2R acts as a carrier protein forIGF-II in the circulatory system, regulating its bioavail-ability and activity [21]. Many of IGF2R’s functions areattributed to its role in regulating the levels of IGF-IIavailable to bind IGF-1R. However, some studies haveidentified IGF-II-stimulated actions that occur via IGF2Ronly (often using the Tyr27Leu IGF-II analogue that can-not bind either IGF-1R or IR-A). These IGF-1R-indepen-dent actions include IGF2R-mediated mitogen activatedprotein kinase (MAPK) pathway activation [17,22], apop-tosis and hypertrophy in cardiomyoblasts [23], andmigration of extravillous trophoblasts [17].

acidic environment. Proteins targeted to lysosomes are degraded and/or

recycled to the plasma membrane.

d. All rights reserved. doi:10.1016/j.tibs.2009.07.003 Available online 30 September 2009

Box 1. Overview of IGF-II and IGFBP structures

The IGF-II structure

IGFs belong to the superfamily of insulin-related proteins that

includes insulin, IGF-I, IGF-II and the relaxins. IGFs consist of so-

called B, C, A and D ‘domains’ (regions of sequence in order from

the N to the C terminus). The B and A domains are similar in

structure to the equivalent domains in insulin. These domains

contain three alpha-helices: Helix 1 (Gly10-Val20 of IGF-II) is in the B

domain, whereas helix 2 (Glu44-Phe48 of IGF-II) and helix 3 (Ala54-

Tyr59 of IGF-II) are both located in the A domain. The C and D

domains are highly flexible. The structure is held together by 3

disulphide bonds (Cys9-Cys47, Cys21-Cys60, Cys46-Cys51). Major

determinants for IGF-1R and IR-A binding are located at the end of

the B domain and the beginning of the A domain (including residues

Val43, Phe26 and Tyr27). Further residues required to achieve high

affinity binding form a second binding site involving residues Glu12,

Phe19, Leu53 and Glu57 [80].

IGFBP structures

IGFBPs share a common domain organisation comprising cysteine-

rich N and C domains connected by a flexible linker. No structure is

available for an intact IGFBP; however, structures of individual N

and C domains have been solved for several IGFBPs, and a crystal

structure of a ternary complex between IGF-I and the individual N

and C domains of IGFBP-4 has been reported. The N domain forms

an ‘‘L’’ shape and contains mainly b-sheet plus some a-helical

content. The C domains adopt a thyroglobulin type-1 fold, with an

initial a-helix followed by a 3-stranded antiparallel b-sheet. Within

this family, IGFBP-6 is unusual as it preferably binds IGF-II (�20-100

fold higher affinity than for IGF-I). The amino acids responsible for

conferring this IGF-II binding specificity have not been identified,

but are believed to reside in the IGFBP-6 C-domain.

Review Trends in Biochemical Sciences Vol.34 No.12

IGF2R knockout in mice results in a lethal phenotypeowing to lung malformation and subsequent respiratoryproblems at birth [24,25]. IGF2R knockouts are rescued bythe simultaneous inactivation of either IGF2 or IGF1R[26]. By contrast, tissue-specific IGF2R knockout inskeletal muscle and liver in adult mice has no effect onphenotype, thus highlighting the importance of IGF2R infoetal development [27]. Like IGF2, IGF2R is also animprinted gene. Single nucleotide polymorphisms (SNPs)in IGF2R are associated with perturbed growth control.Increased birth weight is associated with SNPs that resultin a Leu252Val substitution in Japanese populations[28,29], and a decreased growth rate in the first 3 yearsof life is associated with a resultant Gly1619Arg substi-tution [30]. Somewhat surprisingly, however, theGly1619Arg substitution does not alter IGF-II binding,suggesting that the polymorphism giving rise to thischange might be in linkage disequilibrium with anotherSNP(s) [31].

IGF2R is often referred to as a tumour suppressor gene[32]. SNPs within IGF2R have been associated with anincreased risk of cancer [32,33], and microsatelliteinstability within the gene occurs in a large number ofgastrointestinal cancers [34]. Loss of heterozygosity(LOH) of IGF2R and somatic mutations in the remainingallele are associated with several cancers, including hepa-tocellular carcinoma, gastrointestinal and prostate can-cers [35–38].

There are 6 structurally related, high affinity IGFbinding proteins (IGFBPs; IGFBP-1 to -6) [39,40] (Box1). Most of the actions of IGFBPs described to date involveinteractions with IGFs, although IGFBPs can also act

independently of IGFs (reviewed in [41]). Nearly all circu-lating IGFs are bound to IGFBPs, mostly within theIGFBP-3-acid labile subunit (ALS) complex. PlasmaIGFBPs increase the half-life of circulating IGF; they alsodeliver IGF to tissues [40], where they can either inhibit orpotentiate IGF action by regulating IGF–IGF-1R binding.IGF can be released through proteolysis of IGFBPs [42,43]or by binding of IGFBPs to the extracellular matrix [40].Once released, IGF is available to bind and activate IGF-1R. In mice, the loss of an IGFBP-4 protease, pregnancyassociated plasma protein-A (PAPP-A), leads to prenatalgrowth deficiency, because non-proteolyzed IGFBP-4 can-not release IGF-II as in normal development [44].

Thus, normal growth is achieved by fine control of IGF–

IGF-1R binding, through a balance of IGF-I, IGF-II, IGFBPand IGF2R levels; perturbation of this balance leads todisease. With the recent publication of the structure of theIGF2R–IGF-II complex, it is timely to review the lessonslearned from understanding how the structures of IGFsystem components (IGF-II, IGF2R and IGFBPs) influenceIGF-II function in growth and development.

Molecular mechanisms underlying the control ofIGF-II actionThe structure of IGF-II was determined in the mid 1990s(Box 1) [45]. More recently, significant advances have beenmade in the determination of the structures of the proteinsthat regulate IGF-II action. Not only did we recentlydetermine the IGF2R–IGF-II complex structure [46], butthe structures of several IGFBP subdomains have alsobeen reported [39,47,48]. A comparison of the complexesthat these proteins form with IGF-II reveals commonfeatures that reflect a similar mechanism of IGF-II control.

The IGF2R structure

IGF2R consists of a large N-terminal extracellular region,a single membrane-spanning region and a small cyto-plasmic tail [16]. The extracellular region comprises 15homologous domains [49] that are similar in sequence tothe single extracellular domain of the cation-dependentmannose-6-phosphate receptor (CD-MPR) [50]. Based onthe known structures of domains 1-3 of the bovine protein[50,51] and domains 11–14 of the human protein[16,52,53], each of the 15 IGF2R domains is expected toadopt similar structures (as described for domain 11). Alldomains are predicted to have four equivalent disulphidebonds, with the exception of domains 5, 7 and 15, whichhave only three, and domain 13, which has an extra twodisulphide bonds in a 48-residue fibronectin type II (FnII)insert. This FnII insert projects from domain 13 andnestles close to the IGF-II binding site in domain 11,interfacing with both domains 11 and 12 [46].

There is no intrinsic kinase activity within the IGF2Rcytoplasmic domain, and the receptor is consequentlyregarded as non-signalling. However, the cytoplasmicdomains of both the IGF2R and the CD-MPR containrecognition motifs for assembly proteins 1 and 2, whichregulate their internalization and sorting [13]. Phosphoryl-ation of the cytoplasmic domain has been reported, andsequencemotifs that could potentially act as protein kinasesubstrates have been identified [54]. These features might

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Figure 1. The IGF2R domain 11 and the IGF-II binding site. The structure of the

IGF2R domain 11–IGF-II complex reveals a hydrophobic interface with key residues

conserved across evolution. (a) Ribbon diagram coloured from blue at the N-

terminus to red at the C-terminus. IGF-II (magenta) contacts the IGF2R domain 11

AB, CD, EF and HI loops, with the IGF-II Phe19 residue (shown in stick format)

acting as an anchor. IGF2R domains 12 and 13 from the complex structure are not

shown, because neither makes direct contact with IGF-II. (b) Surface representation

showing residues of the IGF2R domain 11 undergoing a change in accessible

surface area upon ligand binding [52] (depicted in pale yellow). (c) as (b), but with

interface residues that differ in sequence between human and both monotremes

and chicken highlighted in green. Monotreme and chicken IGF2R do not bind IGF-

II; therefore these residues are likely to be important for IGF-II binding by human

IGF2R [62].

Figure 2. The IGF2R–IGF-II interface. The IGF2R domain 11-13–IGF-II complex

structure reveals the relative contribution of each domain for binding IGF-II,

depicted in magenta. (a) Cartoon representation of the IGF2R domain 11-13–IGF-II

complex, with transparent surface representation. The FnII insert (black) in IGF2R

domain 13 (light blue) contributes to the overall affinity of the interaction by

maintaining the domain 11 (yellow) IGF-II binding site in a high-affinity state prior

to ligand binding. IGF2R domain 12 (red) acts as a scaffold within this IGF-II

binding complex. (b) Expanded open-book view of the IGF2R domain 11–IGF-II

interface with side chains undergoing a change in accessible surface area upon

complexation shown as sticks. IGF2R domains 12 and 13 from the complex

structure are not shown.

Review Trends in Biochemical Sciences Vol.34 No.12

play a role in receptor trafficking and, therefore, in IGF2Rfunction [54]. Furthermore, whereas several studiessuggest a role for IGF-II-stimulated IGF2R-dependentactivation of G-protein coupled receptor (GPCR) signalling,the evidence for an association of the IGF2R cytoplasmicdomain with G-proteins is conflicting [16]. Indeed, recentstudies suggest that GPCRs are indirectly activated viaIGF2R following IGF-II-mediated sphingosine-1 phos-phate production [54].

IGF-II binds IGF2R domain 11 [55,56], but high affinitybinding (10-10M) is achieved only in the presence of domain13 [57]. A single amino acid substitution in domain 11 atresidue 1572 (Thr to Ile) completely abrogates IGF-IIbinding [58,59]. The core IGF2R domain structure is aflattened b-barrel comprising nine b-strands formingtwo crossed b-sheets (Figure 1a). Four disulphide bondscontribute to fold stability. At the N-terminus a b-hairpincaps the b-barrel; in the CD-MPR structure this region is a-helical. The domain 11 structure reveals 4 loops (AB, CD,EF, HI) at one end of the b-barrel; these were predicted toform the IGF-II-binding region (Figure 1b) because theyare equivalent spatially to the carbohydrate-bindingpocket of CD-MPR [50,52].

A recent structure of a complex of IGF-II bound to anIGF2R fragment comprising domains 11-13 reveals detailsof the mechanism of IGF-II–IGF2R binding (Figure 2) [46].As predicted, the domain 11 IGF-II binding site involvesthe four previously implicated loops [46]. The resolution ofthe complex structure (4.1 A) allows the generation of a

614

good three dimensional representation of the protein–

protein interaction, but makes it difficult to assign specificside chain contacts between IGF2R and IGF-II. This crys-tal structure, however, disproved two previously generatedmolecular models of the complex [60,61]. Detailed muta-genesis of IGF2R domain 11 residues identified a hydro-phobic cluster consisting of Tyr1542, Phe1567 andLeu1629, which forms the core of the IGF-II binding site[62]. The crystal structure reveals that this core surroundsthe IGF-II anchor residue, Phe19 [46] (Figure 2). Severalresidues in the core are conserved in eutherian mammalsbut not in monotremes and birds, thus explaining the lackof IGF-II–IGF2R binding in these species (Figure 1c andFigure 3). Interestingly the IGF-II residues critical forIGF2R binding are conserved acrossmost species includingchicken and monotremes, leading to the conclusion thatIGF2R evolved to bind IGF-II with the appearance ofeutherian species [16].

The IGF-II–IGF2R crystal structure also reveals thatdomain 13 (including the FnII insert) does not directly

Figure 3. Sequence alignment of IGF2R domain 11 residues. Numbering is based on the human sequence (dots above every 10th residue). Residues conserved across all

species are boxed in blue, whereas residues highlighted in bold are conserved across a number of species and conserved cysteines are indicated by green triangles. Those

residues buried in the IGF2R–IGF-II interface (a change in accessible surface area of > 2 A2 upon binding [46]) are highlighted by gold triangles. Interface residues that are

different from the human in both the monotreme and the chicken sequences and are therefore likely to contribute to the lack of IGF-II binding are indicated by red triangles.

Accession numbers for the sequences are human (Homo sapiens) NP_000867 , cow (Bos taurus) P08169, mouse (Mus musculus) NP_034645 , kangaroo (Macropus

rufogriseus) AAK71865, opossum (Monodelphis domestica) XP_001371436 , platypus (Ornithorhynchus anatingi) AAF68173 , echidna (Tachyglossus aculeatus) AAL23910 ,

chicken (Gallus gallus) NP_990301 , zebrafish (Danio rerio) NP_001034716 .

Review Trends in Biochemical Sciences Vol.34 No.12

contact IGF-II, but rather plays a stabilizing role, main-taining the domain 11 IGF-II binding site in a high-affinitystate prior to ligand binding. A comparable effect isachieved by a Glu1544Lys substitution within isolated

Figure 4. Model of overall IGF2R structure. The possible arrangement of IGF2R domains

IGF2R domains 1-3 and IGF2R domains 11-13. (a) A putative IGF2R monomer, coloured

magenta and mannose-6-phosphate-binding sites shown as pink spheres. Ellipses indi

dimer based on crystal packing observations and biophysical data for IGF2R fragment s

and the other in gray-blue. The monomers contact each other at various points alo

representation of the cell membrane lipid bilayer is shown at the bottom of the figure.

domain 11; this increases the affinity for IGF-II by six-fold[62]. It is possible that the Glu introduced at residue 1544makes an electrostatic interaction with IGF-II Asp20, thusholding the normally flexible AB loop in a favourable

into the final structure is presented in this model using the known structures of the

from blue at the N-terminus to red at the C-terminus, with FnII in black, IGF-II in

cate regions for which X-ray crystallography structures exist. (b) A tentative IGF2R

tructures. Two views are shown. One monomer of each dimer is coloured as in (a)

ng their lengths, in accordance with experimental and crystal packing data. A

This figure is reproduced with permission from Brown et al., 2008 [46].

615

Figure 5. Overlapping binding sites on IGF-II. A comparison of binding sites for

receptors and IGFBPs on IGF-II reveals that they are overlapping. These data

reveal a mechanism by which IGF-II bioavailability is controlled. (a) IGF-II is

depicted as a magenta ribbon with Phe19 as sticks in the two orientations used in

(b)–(d). Binding sites on IGF-II for the IGF2R (b), IGFBPs (c) and IGF-1R (d) have

been mapped by site-directed mutagenesis studies and are highlighted with

dotted ellipses. The residues essential for IGF2R binding correspond with

residues that are also important for IGFBP and IGF-1R binding. This overlap

suggests an evolutionary selection for a region to control interaction with the

IGF-1R, and thus to control IGF-II action. The relative effect of mutations on

binding is shown; � 2-fold decrease = dark blue, > 2 < 6-fold decrease = orange,

� 6-fold decrease = red, �2-fold increase = light blue, not tested = gray. Figure

generated using PDB file 1IGL.

Review Trends in Biochemical Sciences Vol.34 No.12

conformation for binding. Thus, domains 11 and 13 act inconcert to form the high-affinity IGF-II binding site, anddomain 12 appears to act as a scaffold.

Domains 11-14 form a dimer within the crystal lattice;domain 12 plays a significant role in the formation of thisdimer. Ligand-free IGF2R homodimers can be observed onthe cell surface [63,64], although in detergent solutions themost common form is monomeric [65]. It appears thatmultiple interactions between monomers occur along theextracellular domains; domain 12 is particularly important[63,64], but isolated bovine domain 5 can also dimerize[66]. The combination of the known IGF2R crystal struc-tures, along with existing biochemical data allowed amodel for an IGF2R ectodomain monomer and dimer tobe proposed [46] (Figure 4). The tri-domain units equival-ent to domains 1-3 are considered as ‘carbohydrate bindingunits,’ with three copies comprising domains 1-9 (domains3, 5 and 9 are known to bind mannose-6-phosphate). Thestructure of domains 11-14, in the form of its crystallo-graphic dimer, forms the basis for an IGF2R dimer;additional dimerization interactions form between eachmonomer’s domain 5. At this stage, there is no evidenceto suggest that IGF2R dimerization plays a role in IGF-IIbinding or control of IGF-II activity; however, these pos-sibilities have not been fully explored. IGF2R dimerizationmight, however, be more likely to occur when bindingmultivalent mannose-6-phosphate-containing proteins.

The binding sites for mannose-6-phosphate-containingproteins and other ligands are quite distinct from the IGF-II binding site (Figure 4). Two high-affinity binding sitesfor lysosomal enzymes and other mannose-6-phosphate-containing ligands are located in domains 3 and 9, and onelow-affinity site is found in domain 5 [16,66,67]. Thesedomains lack the analogous CD-MPR residue Asp103 thatcoordinates divalent cations to enhance ligand binding[68]. Binding sites for urokinase plasminogen activatorreceptor (uPAR) and plasminogen are located withinIGFR2 domain 1 [51,69–71]. The plasminogen binding siteis located on the opposite face of the domain 1-3 structureto the oligosaccharide binding site and it is thereforepossible to bind simultaneously both mannose-6-phos-phate and uPAR or plasminogen [51]. TGF-b is likely tobind via its mannose-6-phosphate components [72]. Theseobservations have led to the suggestion that IGF2R func-tions to bring together different molecules important forregulating migration and apoptosis.

A role for IGF2R in controlling the activation of latentTGF-b has been proposed, because IGF2R binds bothplasminogen and uPAR [70,72–74]. The urokinase boundto uPAR converts plasminogen to plasmin, which in turnactivates latent TGF-b. Active TGF-b then stimulatesapoptosis via TGF-b receptors, and this activity is pro-posed to influence cell migration and invasion [70,72–74].

The IGF2R binding site on IGF-II

The IGF2R binding site on IGF-II comprises the hydro-phobic residues Leu8, Phe19 and Leu53, together withneighbouring residues Glu6, Thr16 and Asp52 (Figure 2)[46]. These residues are buried in the binding interfacedefined in the crystal structure of the complex [46]. Otherside chains, including Ala54 and Leu55, also play a role in

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IGF2R binding [46,75]. In contrast to earlier reports, thecrystal structure shows that the Phe48, Arg49, Ser50 motifof IGF-II is not directly involved in IGF2R binding [46,76].Although IGF-II shares over 75% sequence identity withIGF-I, and they are structurally very similar, IGF-I isunable to bind IGF2R. We have been unable to detectIGF-I–IGF2R binding, using surface plasmon resonance[77]. It was believed initially that the specificity for IGF-IIresulted from amino acids in the most divergent regionsbetween IGF-I and IGF-II. Interestingly, however, site-directed mutagenesis revealed that a single amino acidburied in the interface with IGF2R is responsible for thebinding specificity for IGF-II and not IGF-I. A substitutionof IGF-II Thr16 to Ala (the equivalent residue in IGF-I)essentially abrogates IGF2R binding, whereas substi-tution of the equivalent IGF-I Ala13 to Thr introducesthe ability to bind IGF2R, albeit with weak affinity [46].

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The IGFBP binding site on IGF-IIThe IGFBPs bind IGF-I and IGF-II with nanomolar affi-nities, but with varying specificities. For example, IGFBPs1 and 4 have equal affinity for both ligands, whereasIGFBP-2 (2-10 fold) and IGFBP-6 (20-100 fold) have ahigher affinity for IGF-II [40]. High affinity binding isachieved through interactions with both the N- and C-terminal domains of the IGFBPs, with the greatest con-tribution stemming from the N-domain. In the case ofIGFBP-6, however, specificity for IGF-II probably lies indeterminants of its C-domain [39]. However, the precisemechanisms responsible for the IGF-II preference have notbeen determined. The residues on IGF-II involved inIGFBP binding have been characterized well by site-directed mutagenesis and structural studies [78](Figure 5). Interestingly, several residues important forIGFBP N-domain binding, including Glu6 and Phe19 [79],are also important for IGF2R binding (Figure 5). As is thecase for the IGF-II–IGF2R complex, the IGF–IGFBP inter-face involves predominantly hydrophobic interactions.IGF-II is unable to bind the IGF-1R when bound to eitherthe IGF2R or IGFBPs, as both complexes involve residuesthat are also important for IGF-1R binding (Figure 5). Itis remarkable that these two structurally distinct high-affinity IGF binding protein families (the IGFBPs and theIGF2R) have evolved to target the same region of IGF-II toprovide tight control of its bioavailability [46]. Presumably,IGF-II–IGFBP binding prevents binding to the IGF2R, andthis in turn could potentially influence any IGF2R-depend-ent IGF-II actions (e.g. MAPK activation). This possibility,however, has not been explored experimentally.

Concluding remarks and future perspectivesA comprehensive definition of the IGF-II–IGFR2 bindinginteraction provides an understanding of the mechanismsby which IGFR2 regulates IGF-II activity. AlthoughIGF2R–IGF-II complex internalization and lysosomaldegradation of IGF-II is the predominant mechanism bywhich IGF2R controls IGF-II actions, several reportssuggest that stimulation of cells with IGF-II leads toIGF2R-dependent activation of MAPK-mediated G-protein-coupled signalling [17,22,23,54]. However, this sig-nalling is not likely to result from a direct interaction of theIGF2R with signalling molecules. A more likely expla-nation is that IGF-II binding influences IGF2R inter-actions with other ligands (perhaps by increasingreceptor turnover upon IGF-II binding), which, in turn,alters biological activity. This is possibly the case whenIGF-II inhibits IGF2R–125I b-Galactosidase binding,because each bind disparate regions of the receptor [16].It is worth noting, however, that binding of latent TGF-b topurified IGF2R can preclude binding of IGF-II, despite thefact that these molecules bind separate sites on the IGF2R[31]. TGF-b binding presumably induces a structuralchange in the IGF2R that sterically occludes IGF-II bind-ing. Such an activity would suggest, under conditions ofhigh latent TGF-b concentration, TGF-b binding (andtherefore activation) would be favoured (i.e. in metastaticbreast cancer, where TGF-b levels are elevated). In sum-mary, several possible mechanisms by which IGF2R influ-ences IGF-II biological activity have been proposed.

However, a comprehensive understanding of how IGF-IIinfluences the binding of other IGF2R ligands and theirsubsequent biological activity will require further investi-gation.

The biological roles of IGF-II in humans are yet to befully elucidated, in part because circulating IGF-II levelsdiffer between humans andmodel organisms such as mice.Invaluable information has been gained by comparing,across species, sequences that dictate IGF2R–IGF-II bind-ing. IGF2R can bind IGF-II only in eutherian mammals,indicating that IGF2R evolved to bind IGF-II with theadvent of placentation. It should be noted that IGF-IIsequences that are important for IGF2R binding have beenconserved in chickens and monotremes. We, therefore,would expect IGF-II from these species to bind eutherianIGF2Rs; however this has not been reported [16]. Suchobservations provide crucial information regarding IGF-IIfunction. Indeed, the large body of genetic, biochemical andbiophysical research on IGFs and their receptors accumu-lated thus far points to a remarkably intricate systemdesigned to finely balance growth and differentiation, withfascinating sequence and structural considerations provid-ing new perspectives on evolution. Future studies willfocus on how IGF-II influences differentiation processes,the role in neural development, stem cell self renewal andthe role of IGF-II in cancer via the IR-A regulated by theIGF2R and IGFBPs.

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