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The extracellular matrix (ECM) is the material found surrounding the cells in a tissue. One component of the ECM is the elastic fiber, which confers the property of elasticity to its environment. Organs such as the lung, skin and major blood vessels have an abundance of elastic fibers so that they are able to expand and recoil. Elastic fibers are composed of two main components; elastin and microfibrils. Microfibrils are composed primarily of fibrillin-1 and provide a scaffold unto which tropoelastin monomers assemble. Elastic fibers interact with many other proteins in the ECM, one of which is fibulin-5. Based on the severe elastic fiber defects observed in the fibulin-5 null mouse, it was established that fibulin-5 plays an essential role in elastic fiber development. This role may be in the deposition of tropoelastin onto microfibrils and/or in stabilizing the elastic fibers in the extracellular matrix. In the present study, the relationship between fibulin-5 and the elastic fiber was investigated through a number of in vivo and in vitro experiments. To test the hypothesis that fibulin-5 requires the presence of elastin to assemble in the ECM, full-length recombinant fibulin-5 (rF5) was purified from transfected cells and used to make a fibulin-5 antibody. Solid-phase binding assays using rF5 showed that fibulin-5 binds tropoelastin at two sites; the initial portion of the C-terminus and the first calcium-binding epidermal growth factor-like domain at the N-terminus. Immunofluorescence staining of elastin null mouse embryonic fibroblast cultures revealed that fibulin-5 does not require elastin to be present in the ECM in order to assemble. Subsequently, solid-phase binding assays showed that fibulin-5 can bind to the N-terminus of fibrillin-1. To determine if fibulin-5 could exist independent of elastin and/or fibrillin-1 in vivo, an immunohistochemical analysis was conducted on heart, liver, lung, colon, spleen, testis and kidney. All three proteins were co-localized in all organs except in the kidney, where fibrillin-1 was found to independently stain the capillary tufts of the renal corpuscles and renal tubules. Thus, fibulin-5 may be co-regulated with elastin and is not present on elastin-independent microfibrils. Additionally, novel locations of elastic fibers were uncovered in the heart, liver, colon, spleen and testis. Overall, this study provides important insights as to the role of fibulin-5 in elastic fiber structure and assembly and also reveals the complexity in understanding the pathogenesis of diseases involving elastic fiber proteins.