Placental development and imprinting co-evolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes, IGF2 and IGF2R, code for the growth promoter insulin-like growth factor 2 and its binding inhibitor, mannose 6-phosphate/IGF2 receptor, respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes that lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that evolution of this ESE led to the fortuitous acquisition of M6P/IGF2R IGF2 binding that drew IGF2R into parental conflict prior to imprinting, that may have accelerated subsequent affinity maturation. † The sequence of molecular evolutionary events that established placental viviparity, genomic imprinting and parental conflict in mammals remain poorly understood (1) . Genomic imprinting occurs when expression of one allele of a diploid gene is silenced depending on the parent-of-origin, e.g. either from the father or the mother. Parental conflict over the distribution of resources to offspring has been supported by the observation of reciprocal imprinting of genes coding for the growth promoter Insulin-like growth factor 2 (IGF2), and the cation-independent mannose 6-phosphate/ IGF2 receptor (M6P/IGF2R or IGF2R) (2) . IGF2 and IGF2R are two of the approximately 80 genes imprinted in mammals, and two of the five genes (with INS, MEST/PEG1 and PEG10) imprinted in marsupials. So far, no evidence supports the existence of imprinting in monotremes despite the presence of a chorio-vitelline placenta (3, 4). On the basis of functional data, IGF2R transports M6P modified acid hydrolases to the pre-lysosomes (5). Of the 15 extra-cellular domains of IGF2R, domain 11 binds IGF2 in therians, and internalizes the ligand for degradation, whereas M6P bind to domains 3, 5 and 9 (5). Igf2 rescues placental dependent embryonic lethality associated with laboratory created murine parthenogenesis, implicating IGF2 supply as a regulator of placental development (6). Disruption of the maternal Igf2r allele results in Igf2 dependent overgrowth and fatality, supporting that IGF2R antagonizes the function of IGF2 (7,8). The structure of the unbound human domain 11 shows that the IGF2 binding site composed of defined loops (AB, CD, FG and HI, Fig. 1A and Fig. S1) but how this domain 11 evolved to bind IGF2, and the relationship to imprinting co-evolution, remain unknown (9-12).We established a high resolution structure of the human IGF2R:IGF2 complex and then compared this to other phylogenetically informative vertebrates. We adopted an NMR approach as the side chain amino acid interactions across the binding interface were not resolved in our 4.1Å resolution co-crystal structures (9). Wild-type human domain 11 and IGF2 failed to form a stable association in initial NMR studies. However, we ...
The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.
Among the 15 extracellular domains of the mannose 6-phosphate/ insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the k off value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.growth factor receptor | protein evolution | insulin-like growth factor 2 | binding kinetics | biological therapy T he functional evolution of proteins is largely considered to occur by chance, frequently because of unpredictable and specific events that confer a structure-based change in function sufficient for subsequent selection or "gain of fitness" (1). One such evolutionary biochemical example is the initial acquisition and subsequent gain of affinity between the insulin-like growth factor 2 (IGF2) ligand and a single domain of a nonsignaling mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R) (domain 11). The structural and functional basis of this evolutionary path, which has occurred over 150 million years of mammalian evolution, has been reported previously (2). The questions that we address in the present work are whether the IGF2:domain 11 interaction has reached an optimal state in the context of IGF2 activation of signaling receptors and in the ligand clearance function of M6P/IGF2R, and how far can we extend the binding interaction in terms of structural, biophysical, and functional properties.Functionally, and unlike products of other mammalian imprinted genes, domain 11 is unusual because it specifically evolved to bind to an evolutionary conserved IGF2 ligand with high affinity (3-5). After binding, clearance of extracellular IGF2...
The mannose 6-phosphate/IGF 2 receptor (IGF2R) is comprised of 15 extra-cellular domains that bind IGF2 and mannose 6-phosphate ligands. IGF2R transports ligands from the Golgi to the pre-lysosomal compartment and thereafter to and from the cell surface. IGF2R regulates growth, placental development, tumour suppression and signalling. The ligand IGF2 is implicated in the growth phenotype, where IGF2R normally limits bioavailability, such that loss and gain of IGF2R results in increased and reduced growth respectively. The IGF2R exon 34 (5002A>G) polymorphism (rs629849) of the IGF2 specific binding domain has been correlated with impaired childhood growth (A/A homozygotes). We evaluated the function of the Gly1619Arg non-synonymous amino acid modification of domain 11. NMR and X-ray crystallography structures located 1619 remote from the ligand binding region of domain 11. Arg1619 was located close to the fibronectin type II (FnII) domain of domain 13, previously implicated as a modifier of IGF2 ligand binding through indirect interaction with the AB loop of the binding cleft. However, comparison of binding kinetics of IGF2R, Gly1619 and Arg1619 to either IGF2 or mannose 6-phosphate revealed no differences in ‘on’ and ‘off’ rates. Quantitative PCR, 35S pulse chase and flow cytometry failed to demonstrate altered gene expression, protein half-life and cell membrane distribution, suggesting the polymorphism had no direct effect on receptor function. Intronic polymorphisms were identified which may be in linkage disequilibrium with rs629849 in certain populations. Other potential IGF2R polymorphisms may account for the correlation with childhood growth, warranting further functional evaluation.
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