Abstract
The cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R or IGF2R) traffics IGF2 and M6P ligands between pre-lysosomal and extra-cellular compartments. Specific IGF2 and M6P high-affinity binding occurs via domain-11 and domains-3-5-9, respectively. Mammalian maternal Igf2r allele expression exceeds the paternal allele due to imprinting (silencing). Igf2r null-allele maternal transmission results in placenta and heart over-growth and perinatal lethality (>90%) due to raised extra-cellular IGF2 secondary to impaired ligand clearance. It remains unknown if the phenotype is due to either ligand alone, or to both ligands. Here, we evaluate Igf2r specific loss-of-function of the domain-11 IGF2 binding site by replacing isoleucine with alanine in the CD loop (exon 34, I1565A), a mutation also detected in cancers. Igf2rI1565A/+p maternal transmission (heterozygote), resulted in placental and embryonic over-growth with reduced neonatal lethality (<60%), and long-term survival. The perinatal mortality (>80%) observed in homozygotes (Igf2rI1565A/I1565A) suggested that wild-type paternal allele expression attenuates the heterozygote phenotype. To evaluate Igf2r tumour suppressor function, we utilised intestinal adenoma models known to be Igf2 dependent. Bi-allelic Igf2r expression suppressed intestinal adenoma (ApcMin). Igf2rI1565A/+p in a conditional model (Lgr5-Cre, Apcloxp/loxp) resulted in worse survival and increased adenoma proliferation. Growth, survival and intestinal adenoma appear dependent on IGF2R-domain-11 IGF2 binding.
Highlights
The function of the fifteen homologous extra-cellular domains of the cation-independent mannose 6-phosphate/ insulin-like growth factor 2 receptor (M6P/IGF2R or IGF2R) include the binding, trafficking and extra-cellular internalisation of ligands, such as Insulin-like growth factor 2 (IGF2), mannose 6-phosphate (M6P) modified lysosomal proteases and plasminogen[1]
To date there have been no specific point mutations introduced into the mouse germ-line that result in specific loss of function of Igf2r protein domains
The loss of function of Igf2r maternal expressed allele in mouse development suggest that both loss of mannose 6-phosphate binding and lysosomal enzyme supply may contribute to the disproportionate growth phenotype, as the mechanism of growth regulation of Igf[2] appears proportionate to the mechanisms of cell proliferation and cell death at embryonic day 932
Summary
The function of the fifteen homologous extra-cellular domains of the cation-independent mannose 6-phosphate/ insulin-like growth factor 2 receptor (M6P/IGF2R or IGF2R) include the binding, trafficking and extra-cellular internalisation of ligands, such as Insulin-like growth factor 2 (IGF2), mannose 6-phosphate (M6P) modified lysosomal proteases and plasminogen[1]. For the first time, we introduce a direct mouse knock-in mutation of one of the IGF2 binding residues in the binding site CD-loop of domain 11, that results in replacement of isoleucine, a hydrophobic amino acid,with alanine (I15721 in human, I1565 in mouse) This mutation has been detected in human liver cancer[28], along with other loss-of-function mechanisms such as expansion of a polyG tract in colorectal cancer. It has been proposed that tumour growth promotion might result because of increased local IGF2 supply, as a result of the specific loss of IGF2 binding to IGF2R The phenotype of this single knock-in allele mutation in the mouse (Igf2rI1565A) resulted in a placental and embryonic over-growth phenotype and partial neonatal lethality, with at least 40% of heterozygous mice surviving into adulthood. As a result of this attenuated phenotype, we were able to further characterise the tumour suppressor function using this novel Igf2rI1565A allele in adult intestinal adenoma models
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