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Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

Hughes J, Frago S, Bühnemann C, Carter EJ, Hassan AB - PLoS ONE (2013)

Bottom Line: Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality.In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r.This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Tumour Growth Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

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Surface Plasmon resonance binding kinetics of IGF2 binding to human IGF2R, and mass spectrometry (LCMS) of immune-precipitated liver IGF2R from control Igf2r+m/+p and homozygote transmission of Igf2r+HUm/HUp.Immobilised human IGF2R on a biosensor chip were bound to either recombinant human (A) or mouse (B) IGF2 at increasing concentrations, and real time kinetics fitted to a 1∶1 binding model. (C) steady state binding kinetics were also calculated for both interactions and tabulated. Mouse IGF2 has slightly lower KD (nM). (D) peptides identified by mass spectrometry in samples of immuno-precipitated Igf2r+m/+p (WT) and Igf2rHUm/HUp. In both samples, red boxes depict unique peptides matching mouse IGF2R protein sequence, green boxes depict unique peptides matching human IGF2R protein sequence, and yellow boxes depict areas of overlap between mouse and human peptides. Blue boxes depict peptides common to both sequences. Exons 1–4 are represented by upper numbers, lower numbers represent amino acid residue number. Both mouse and human peptides were detectable in suggesting trans-splicing and generation of mouse and humanised proteins from the humanised alleles.
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pone-0057270-g007: Surface Plasmon resonance binding kinetics of IGF2 binding to human IGF2R, and mass spectrometry (LCMS) of immune-precipitated liver IGF2R from control Igf2r+m/+p and homozygote transmission of Igf2r+HUm/HUp.Immobilised human IGF2R on a biosensor chip were bound to either recombinant human (A) or mouse (B) IGF2 at increasing concentrations, and real time kinetics fitted to a 1∶1 binding model. (C) steady state binding kinetics were also calculated for both interactions and tabulated. Mouse IGF2 has slightly lower KD (nM). (D) peptides identified by mass spectrometry in samples of immuno-precipitated Igf2r+m/+p (WT) and Igf2rHUm/HUp. In both samples, red boxes depict unique peptides matching mouse IGF2R protein sequence, green boxes depict unique peptides matching human IGF2R protein sequence, and yellow boxes depict areas of overlap between mouse and human peptides. Blue boxes depict peptides common to both sequences. Exons 1–4 are represented by upper numbers, lower numbers represent amino acid residue number. Both mouse and human peptides were detectable in suggesting trans-splicing and generation of mouse and humanised proteins from the humanised alleles.

Mentions: It was possible that the humanised allele associated overgrowth may have also related to changes in relative affinity of human IGF2R to mouse IGF2. In order to address this possibility, we expressed and purified the extra-cellular domains (domains 1–15) of recombinant human IGF2R and performed surface plasmon resonance to determine relative affinities to human and mouse IGF2. We show that the affinity (KD) of recombinant human IGF2R for mouse IGF2 is high between 3–6 nM, but slightly lower than that of human IGF2 (1–4 nM) (Fig. 7A, B and C).


Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

Hughes J, Frago S, Bühnemann C, Carter EJ, Hassan AB - PLoS ONE (2013)

Surface Plasmon resonance binding kinetics of IGF2 binding to human IGF2R, and mass spectrometry (LCMS) of immune-precipitated liver IGF2R from control Igf2r+m/+p and homozygote transmission of Igf2r+HUm/HUp.Immobilised human IGF2R on a biosensor chip were bound to either recombinant human (A) or mouse (B) IGF2 at increasing concentrations, and real time kinetics fitted to a 1∶1 binding model. (C) steady state binding kinetics were also calculated for both interactions and tabulated. Mouse IGF2 has slightly lower KD (nM). (D) peptides identified by mass spectrometry in samples of immuno-precipitated Igf2r+m/+p (WT) and Igf2rHUm/HUp. In both samples, red boxes depict unique peptides matching mouse IGF2R protein sequence, green boxes depict unique peptides matching human IGF2R protein sequence, and yellow boxes depict areas of overlap between mouse and human peptides. Blue boxes depict peptides common to both sequences. Exons 1–4 are represented by upper numbers, lower numbers represent amino acid residue number. Both mouse and human peptides were detectable in suggesting trans-splicing and generation of mouse and humanised proteins from the humanised alleles.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585325&req=5

pone-0057270-g007: Surface Plasmon resonance binding kinetics of IGF2 binding to human IGF2R, and mass spectrometry (LCMS) of immune-precipitated liver IGF2R from control Igf2r+m/+p and homozygote transmission of Igf2r+HUm/HUp.Immobilised human IGF2R on a biosensor chip were bound to either recombinant human (A) or mouse (B) IGF2 at increasing concentrations, and real time kinetics fitted to a 1∶1 binding model. (C) steady state binding kinetics were also calculated for both interactions and tabulated. Mouse IGF2 has slightly lower KD (nM). (D) peptides identified by mass spectrometry in samples of immuno-precipitated Igf2r+m/+p (WT) and Igf2rHUm/HUp. In both samples, red boxes depict unique peptides matching mouse IGF2R protein sequence, green boxes depict unique peptides matching human IGF2R protein sequence, and yellow boxes depict areas of overlap between mouse and human peptides. Blue boxes depict peptides common to both sequences. Exons 1–4 are represented by upper numbers, lower numbers represent amino acid residue number. Both mouse and human peptides were detectable in suggesting trans-splicing and generation of mouse and humanised proteins from the humanised alleles.
Mentions: It was possible that the humanised allele associated overgrowth may have also related to changes in relative affinity of human IGF2R to mouse IGF2. In order to address this possibility, we expressed and purified the extra-cellular domains (domains 1–15) of recombinant human IGF2R and performed surface plasmon resonance to determine relative affinities to human and mouse IGF2. We show that the affinity (KD) of recombinant human IGF2R for mouse IGF2 is high between 3–6 nM, but slightly lower than that of human IGF2 (1–4 nM) (Fig. 7A, B and C).

Bottom Line: Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality.In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r.This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Tumour Growth Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

Show MeSH
Related in: MedlinePlus