Systematic mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain - An acidic cluster containing a key aspartate is important for function in lysosomal enzyme sorting

We have used systematic mutational analysis to identify signals in the 166-residue murine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain required for efficient sorting of lysosomal enzymes. Alanine cluster mutagenesis on all conserved residues apart from the endocytosis signal demonstrates that the major sorting determinant is a conserved casein kinase II site followed by a dileucine motif (157DDSDEDLL164). Small deletions or additions outside this region have severe to mild effects, indicating that context is important. Single residue mutagenesis indicates that cycles of serine phosphorylation/dephosphorylation are not obligatory for sorting. In addition, the two leucine residues and four of the five negatively charged residues can readily tolerate conservative substitutions. In contrast, aspartate 160 could not tolerate isoelectric or isosteric substitutions, implicating it as a critical component of the sorting signal

Characterization of the Dimerization Domains on the Mannose-6-phosphate/Insulin-like Growth Factor II Receptor

The mannose-6-phosphate/insulin-like growth factor II (M6P/IGF2) receptor is a transmembrane protein known to sequester growth factors from the extracellular matrix. This behavior suggests a mechanism of tumor suppression. Structurally, the receptor’s extracellular region is segmented into 15 homologous repeats, which are divided further into 5 triplet domains, labelled 1-3, 4-6, 7-9, 10-12, and 13-15. What is notable about the triplets is their propensity to form dimers with triplets on a second M6P/IGF2 receptor. In fact, previous studies indicate that this protein functions optimally when dimerized. Thus, the purpose of this experiment is to characterize these domain interactions. Using a urea and dithiothreitol (DTT) disruption assay, the 7-9 triplet’s potential to dimerize was assessed. Preliminary results indicate that proximity is important for mediating interactions. The 7-9 triplet binds strongly to other 7-9 triplets on a separate M6P/IGF2 receptor; however, its association with any other triplet is not as strong comparatively

Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs

Mannose-6-Phosphate/Insulin-Like Growth Factor 2 Receptor (M6P/IGF2-R) in Growth and Disease: A Review

This work aims to summarize the current knowledge about Mannose-6- Phosphate/Insulin-like Growth Factor 2 Receptor (M6P/IGF2-R) in the regulation of growth and development, and its involvement in tumor progression. M6P/IGF2-R binds both molecules sharing M6P signals and IGF2. The studies showed that M6P/IGF2-R is involved in the trafficking of mannnose-6-phosphorylated enzymes from the Trans-Golgi Network (TGN) to lysosomes and the uptake of secreted proenzymes from the plasma membrane to the lysosomes via clathrin-coated vesicles for their maturation. The M6P/IGF2-R acts as a scavenger that binds IGF2 and transports it to lysosomes for its degradation since IGF2 exerts its biological effects on cell proliferation and development by binding with lower affinity on IGF1 receptor, which is structurally similar to insulin receptor and different from the M6P/IGF2-R. The M6P/IGF2-R has also been studied in human cancer, and frequent losses of heterozygosity (LOH) at the 6q25-27 gene region with mutations in the remaining allele have been described. These results led to consider M6P/IGF2-R gene as a putative tumor suppressor and its potential prognostic value has been suggested

Decreased expression of the mannose 6- phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells

BACKGROUND: Loss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies. METHODS: In this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells. RESULTS: Our results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with the ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis. CONCLUSIONS: These results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth

M6P/IGF2R loss of heterozygosity in head and neck cancer associated with poor patient prognosis

BACKGROUND: The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes for a multifunctional receptor involved in lysosomal enzyme trafficking, fetal organogenesis, cytotoxic T cell-induced apoptosis and tumor suppression. The purpose of this investigation was to determine if the M6P/IGF2R tumor suppressor gene is mutated in human head and neck cancer, and if allelic loss is associated with poor patient prognosis. METHODS: M6P/IGF2R loss of heterozygosity in locally advanced squamous cell carcinoma of the head and neck was assessed with six different gene-specific nucleotide polymorphisms. The patients studied were enrolled in a phase 3 trial of twice daily radiotherapy with or without concurrent chemotherapy; median follow-up for surviving patients is 76 months. RESULTS: M6P/IGF2R was polymorphic in 64% (56/87) of patients, and 54% (30/56) of the tumors in these informative patients had loss of heterozygosity. M6P/IGF2R loss of heterozygosity was associated with a significantly reduced 5 year relapse-free survival (23% vs. 69%, p = 0.02), locoregional control (34% vs. 75%, p = 0.03) and cause specific survival (29% vs. 75%, p = 0.02) in the patients treated with radiotherapy alone. Concomitant chemotherapy resulted in a better outcome when compared to radiotherapy alone only in those patients whose tumors had M6P/IGF2R loss of heterozygosity. CONCLUSIONS: This study provides the first evidence that M6P/IGF2R loss of heterozygosity predicts for poor therapeutic outcome in patients treated with radiotherapy alone. Our findings also indicate that head and neck cancer patients with M6P/IGF2R allelic loss benefit most from concurrent chemotherapy

Transcriptional regulation of the IGF signaling pathway by amino acids and insulin-like growth factors during myogenesis in Atlantic salmon

The insulin-like growth factor signalling pathway is an important regulator of skeletal muscle growth. We examined the mRNA expression of components of the insulin-like growth factor (IGF) signalling pathway as well as Fibroblast Growth Factor 2 (FGF2) during maturation of myotubes in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The transcriptional regulation of IGFs and IGFBP expression by amino acids and insulin-like growth factors was also investigated. Proliferation of cells was 15% d(-1) at days 2 and 3 of the culture, increasing to 66% d(-1) at day 6. Three clusters of elevated gene expression were observed during the maturation of the culture associated with mono-nucleic cells (IGFBP5.1 and 5.2, IGFBP-6, IGFBP-rP1, IGFBP-2.2 and IGF-II), the initial proliferation phase (IGF-I, IGFBP-4, FGF2 and IGF-IRb) and terminal differentiation and myotube production (IGF2R, IGF-IRa). In cells starved of amino acids and serum for 72 h, IGF-I mRNA decreased 10-fold which was reversed by amino acid replacement. Addition of IGF-I and amino acids to starved cells resulted in an 18-fold increase in IGF-I mRNA indicating synergistic effects and the activation of additional pathway(s) leading to IGF-I production via a positive feedback mechanism. IGF-II, IGFBP-5.1 and IGFBP-5.2 expression was unchanged in starved cells, but increased with amino acid replacement. Synergistic increases in expression of IGFBP5.2 and IGFBP-4, but not IGFBP5.1 were observed with addition of IGF-I, IGF-II or insulin and amino acids to the medium. IGF-I and IGF-II directly stimulated IGFBP-6 expression, but not when amino acids were present. These findings indicate that amino acids alone are sufficient to stimulate myogenesis in myoblasts and that IGF-I production is controlled by both endocrine and paracrine pathways. A model depicting the transcriptional regulation of the IGF pathway in Atlantic salmon muscle following feeding is proposed.Publisher PDFPeer reviewe

Exploitation of the Ligand-Binding Properties of the Mannose 6-Phosphate/Insulin-Like Growth Factor II (IGF-II) Receptor to Inhibit IGF-II-Dependent Growth of Cancer Cells

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is a multifunctional, type I transmembrane receptor that is a member of the P-type lectin family. A large, extracytoplasmic (EC) region of the M6P/IGF2R binds various ligands, allowing the receptor to regulate multiple biological functions, including the role as a tumor suppressor. Two major classes of ligands, M6P-glycosylated (i.e. any proteins that bear M6P due to post-translational modification in the trans-Golgi network (TGN)) and non-glycosylated (i.e., the mitogen insulin-like growth factor II (IGF-II)), bind within distinct regions of the EC of the receptor and are trafficked to the lysosome. The M6P/IGF2R as well as the cation-dependent mannose 6-phosphate receptor (CD-MPR) are mostly involved in lysosomal biogenesis, trafficking newly synthesized lysosomal enzymes from the TGN to the early endosomes, where the vesicles mature into lysosomes. The receptors undergo recycling during the late endosomal phase where they are retrograde transported back to the TGN for another round of trafficking. However, a fraction of the receptors is found on the cell surface, where the M6P/IGF2R, but not the CD-MPR, is able to bind extracellular ligands. Through this action, IGF-II can bind to the M6P/IGF2R and will be degraded in the lysosome, reducing the bioavailability of the growth factor for the mitogenic insulin-like growth factor I receptor (IGF1R); thus, the M6P/IGF2R is considered a clearance receptor and tumor suppressor. Due to its growth suppressive function, the M6P/IGF2R is believed to play a role in cancer biology. High-affinity, bivalent M6P-based ligands, such as lysosomal enzymes, bind and stabilize the dimeric M6P/IGF2R at the cell surface, leading to its internalization at a faster rate than when there is no M6P-based ligand bound. Therefore, the major goal of our work is to produce a panel of M6P-based ligands capable of bi- or multivalent binding to the M6P/IGF2R that could suppress IGF-II-dependent growth of cancer cells. Additionally, the M6P receptors (MPR) are well conserved through evolution, with the earliest form of “true” MPR known to date in the invertebrates such as mollusk. However, the social amoeba, D. discoideum, produces lysosomal enzymes that bind to the M6P/IGF2R, a discovery that predated identification of a receptor capable of transporting these acid hydrolases within this organism. We provide evidence of a putative MPR protein that retains all the necessary components of a M6P receptor homology domain that also binds M6P. The studies presented herein further our understanding of the origin of the M6P/IGFR as well as exploiting this receptor as a novel therapeutic target against IGF-II-dependent cancers