Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19.
Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 mum diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres((R)), sulfate modified polystyrene filled with a fluorophore) was conducted at 37 degrees C for 1 h using 20 microg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.
Ainscough
S Louise
SL
School of Life Sciences, Queensland University of Technology, Brisbane, 4001, Queensland, Australia. sl.ainscough@qut.edu.au
Feigl
Beatrix
B
Malda
Jos
J
Harkin
Damien G
DG
D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism*; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism; D018969:Insulin-Like Growth Factor Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008863:Microspheres; D010587:Phagocytosis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013329:Structure-Activity Relationship; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
C515497:IGFBP3 protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019096:Vitronectin; D007334:Insulin-Like Growth Factor I; D006493:Heparin