title: Advanced glycation end-product receptor interactions on microvascular cells occur within caveolin-rich membrane domains.
abstract: Advanced glycation end products (AGEs) have an important role in diabetic complications, with many responses mediated through AGE-receptors. The current study has investigated the binding and uptake of AGEs by retinal microvascular endothelium in an attempt to understand the nature of AGE-interaction with receptors on the cell surface. There has been special emphasis placed on the R1, R2, and R3 components of AGE-receptor complex (AGE-RC) and their localization to caveolin-rich membrane domains. Retinal microvascular endothelial cells (RMECs) were exposed to either AGE-modified BSA (AGE-BSA) or native BSA conjugated to colloidal gold (gAGE, gBSA) for various time periods, fixed, and processed for transmission electron microscopy (TEM). Localization of AGE-RC components in caveolae was investigated using confocal microscopy and ultrastructural immunogold labeling. Caveolae were extracted from RMECs using differential Triton X-100 solubility, and Western analysis was conducted to test for caveolae enrichment and the presence of AGE-RC complex components. Ligand blots determined 125I-AGE-BSA binding to caveolae-enriched extracts. Colloidal gold conjugates of AGE-BSA bound to caveolae and were internalized to be trafficked to lysosomal-like compartments. AGE-receptor complex components were significantly enriched within caveolae. The data suggest that AGEs interact with their receptors within caveolae. It is significant that the AGE-R complex localizes to these organelles, because this may have implications for AGE binding, internalization, signal transduction, and the modulation of AGE-receptor-mediated vascular cell dysfunction.
authors.affiliation: Department of Ophthalmology, The Queen's University of Belfast, Northern Ireland, UK. a.stitt@qub.ac.uk
authors.forename: A W
authors.initials: AW
authors.lastname: Stitt
authors.forename: G A
authors.initials: GA
authors.lastname: Burke
authors.forename: F
authors.initials: F
authors.lastname: Chen
authors.forename: C B
authors.initials: CB
authors.lastname: McMullen
authors.forename: H
authors.initials: H
authors.lastname: Vlassara
mesh_terms: D000818:Animals; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D003930:Diabetic Retinopathy / Q000209:etiology; D004730:Endothelium, Vascular / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D008833:Microcirculation; D008954:Models, Biological; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D012171:Retinal Vessels / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D022461:Caveolins; D017127:Glycation End Products, Advanced; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic