| Abstract |
Fras1, Frem1, Frem2 and Frem3, which comprise a group of four structurally correlated proteins, are the members of the Fras1/Frem extracellular matrix protein family. With the exception of Frem3, deficiency in the individual Fras1/Frem genes gives rise to the ‘bleb’ phenotype, the hallmark features of which are embryonic dermal-epidermal detachment and subsequent formation of sub-epidermal blisters, cryptophthalmos, syndactyly and renal defects.
Given Fras1/Frem structural similarities and related phenotypic defects caused by their deficiency, in the present study we concentrated on defining their localization profile during mouse embryogenesis, in order to examine if their analogous structural and functional characteristics are also reflected in their topology. Indeed, they all display a similar localization pattern in the basement membrane of all embryonic epithelia examined. Furthermore, Frem1 displayed an intracellular expression pattern in the periderm cells of the epidermis. In parallel experiments, Collagen VII, the deficiency of which results in postnatal, rather than embryonic dermal-epidermal detachment, is detected in the basal lamina of many embryonic epithelia at late embryonic stages, when Fras1/Frem levels decline. This fact led us to the interesting assumption that collagen VII compensates for the loss of Fras1/Frem in adult life and vice versa.
With a view to elucidate the function of a protein, its interactions with other proteins should be examined. In vivo observations of VEGF loss from epithelial basement membrane structures in Fras1-/- embryos, urged us to examine a possible interaction between Fras1 and VEGF. In this study, for the first time we demonstrate that the first six chordin-type cysteine rich motifs of Fras1 can directly interact with VEGF and determined the regions that are essential for this interaction. For the requirements of this study, mIMCD polarised epithelial cells and the heterologous two hybrid system in Saccharomyces cereviciae were utilized. Given the fact that VEGF plays an essential role in tumour progression, we determined Fras1/Frem localization profile in chemically induced epidermal tumours. The only member of Fras1/Frem protein family that was detected in the basement membrane of neoplastic epithelia and tumour blood vessels of the underlying connective tissue was Frem3 which, as shown by RNA in situ hybridization experiments, is expressed by tumourigenic epithelia.
Besides VEGF, Fras1 can also interact with the cytoplasmic protein Grip1, which targets Fras1 for secretion to the basolateral site of epithelia. Here we show that co-expression of Fras1 and Grip1 in eukaryotic cells results in endoplasmic reticulum reorganization. The ER aggregates at the points of Fras1/Grip1 co-localization. We studied this phenomenon utilizing specific intracellular markers and identified the regions of the two proteins that are causative for this ER relocation. Frem2, another member of the Fras1/Frem protein family, which has been shown to interact in vitro with Grip1, does not cause similar ER rearrangement. Thus, Fras1 appears to be the only Fras1/Frem member implicated so far in the modification of the cellular structure.
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Λειτουργική μελέτη των πρωτεινών της οικογένειας Fras1/Frem σε φυσιολογικές και παθολογικές καταστάσεις
