Abstract 426: The molecular microenvironment critically affects the formation of isoDGR-dependent integrin binding sites in fibronectin

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Fibronectin (FN) is an adhesive protein present in most body fluids and in the extracellular matrix of many tissues. This protein mediates a variety of cellular interactions with extracellular matrix and play important roles in haemostasis, thrombosis, inflammation, wound repair and angiogenesis. FN is constituted of three types of repeating homologous modules termed FN-I, FN-II and FN-III. Each module contains binding sites for molecules of the extracellular matrix and for cell adhesion receptors, such as integrins. Moreover, FN contains four NGR sites located in the: 5th type I, 2nd type II, 7th type I repeat, 9th type III repeats. We have previously shown that the NGR site of the 5th type I repeat (FN-I5) rapidly undergoes deamidation leading to formation of isoDGR. A deamidated FN fragment corresponding to this domain binds αvβ3, inhibits endothelial cell adhesion and proliferation and inhibits tumor growth in animal models. In the present study we have investigated the molecular mechanisms that regulate isoDGR formation in FN in vivo. We provide evidence to suggest that FN purified from plasma contains very low amounts of isoDGR ( 17% in 4 h). In vitro cell adhesion assays, antibody binding assays and integrin binding studies showed that FN deamidation can rapidly occur when FN is bound to microtiter plates, a condition known to cause conformational changes in this protein, but not with FN in solution. Rapid deamidation was observed to occur also with short fragments (FN-I5), either adsorbed on microtiter plates or in solution. These results suggest that the kinetics of NGR-to-isoDGR transition in plasma FN is slower than its clearance rate. Furthermore, the finding that conformational changes and/or protein fragmentation increase the kinetics of NGR-to-isoDGR transition suggest that changes in the molecular microenvironment of NGR sites, potentially occurring after FN deposition in tissues, could be critical for the formation of isoDGR integrin binding sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2011-426