RESUMO
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 koff 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.
Assuntos
Fator de Crescimento Insulin-Like II/metabolismo , Receptor IGF Tipo 2/metabolismo , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Cristalografia por Raios X , Evolução Molecular Direcionada , Humanos , Fator de Crescimento Insulin-Like II/química , Fator de Crescimento Insulin-Like II/genética , Modelos Moleculares , Pichia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptor IGF Tipo 2/antagonistas & inibidores , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/genéticaRESUMO
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, (35)S 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.
Assuntos
Substituição de Aminoácidos , Fator de Crescimento Insulin-Like II/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/genética , Animais , Membrana Celular/metabolismo , Proliferação de Células , Citometria de Fluxo , Meia-Vida , Humanos , Cinética , Ligantes , Desequilíbrio de Ligação/genética , Manosefosfatos/metabolismo , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor IGF Tipo 2/metabolismo , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.
Assuntos
Fator de Crescimento Insulin-Like II/química , Fator de Crescimento Insulin-Like II/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Fator de Crescimento Insulin-Like II/genética , Mutagênese , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Receptor IGF Tipo 2/genética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas/metabolismo , Receptor IGF Tipo 2/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Primers do DNA , Humanos , Fator de Crescimento Insulin-Like II , Dados de Sequência Molecular , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Reação em Cadeia da Polimerase , Ligação Proteica , Conformação Proteica , Proteínas/química , Proteínas/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Ligands transported by the mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor (IGF2R) include IGF-II- and mannose 6-phosphate-modified proteins. Increased extracellular supply of IGF-II, either secondary to loss of the clearance function of IGF2R, loss of IGF binding protein function, or increased IGF2 gene expression, can lead to embryonic overgrowth and cancer promotion. Reduced supply of IGF-II is detrimental to tumor growth, and this suggests that gain of function of IGF-II is a molecular target for human cancer therapy. Domain 11 of IGF2R binds IGF-II with high specificity and affinity. Mutagenesis studies have shown that substitution of glutamic acid for lysine at residue 1554 results in a 6-fold higher affinity for IGF-II (20.5 nmol/L) than native domain 11 (119 nmol/L). Here, we generate a novel high-affinity IGF-II ligand trap by fusion of mutated human 11(E1554K) to a COOH-terminal human IgG1 Fc domain (11(E1554K)-Fc). The resulting homodimer has a significantly increased affinity for IGF-II (1.79 nmol/L) when measured by surface plasmon resonance. IGF-II signaling via the IGF-I receptor and the proliferative effect of IGF-II were specifically inhibited by 11(E1554K)-Fc in both HaCaT and Igf2(-/-) mouse embryonic fibroblast cells. These data confirm that a novel engineered and soluble IGF2R-11(E1554K)-Fc protein functions as an IGF-II-specific and high-affinity ligand trap in vitro and that this protein has potential application as an IGF-II antagonist for cancer therapy following in vivo experimental evaluation.
Assuntos
Proliferação de Células , Fibroblastos/metabolismo , Fator de Crescimento Insulin-Like II/fisiologia , Queratinócitos/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Western Blotting , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero , Fibroblastos/citologia , Vetores Genéticos , Humanos , Fator de Crescimento Insulin-Like II/genética , Ligantes , Camundongos , Camundongos Knockout , Pichia/química , Pichia/metabolismo , Ligação Proteica , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/genética , Receptores Fc/genética , Receptores Fc/metabolismo , Ressonância de Plasmônio de Superfície , Timidina/metabolismoRESUMO
Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.