The essence of this proposal is to study the biological function of the secreted form of APP-751, also called protease nexin II (PNII). Our previous work has demonstrated that APP is involved in the growth regulation of fibroblasts. This tropic activity has been detected for the secreted forms of both APP-695 and APP-751, however, we do not know if they use the same mechanism to exert their respective activity. Our subsequent studies sought to characterize the function of APP-695 because this is a neural-specific isoform of APP, whereas APP-751 is found throughout the organism. We carried out a functional mapping of the molecule and found that the site responsible for the trophic activity observed on the fibroblasts is the pentamer RERMS, from Arg328 to Ser332 of APP-695. Interestingly, this site is within a 17-mer domain which, directly or indirectly, is involved in the binding of APP to heparin. We also found that APP-695 can stimulate the neurite extension of neuroblastoma cells, and that this neurotrophic activity is also mediated through the RERMS sites. However, we do not know whether PNII exerts its function through the same site. It is possible that the presence of the KPI in PNII alters the conformation of the molecule in a way that impairs the accessibility of the RERMS site. This might result in an alteration of the heparin binding properties, and/or in a different biological activity. Therefore, this proposal will focus on these two aspects of APP-751. The heparin binding properties and the ability to stimulate the growth of fibroblasts will be studied for both NEXII (equivalent to PNII, synthesized in bacteria), and NEXII delta, in which the APP-695 active domain is removed. Understanding the physiological function of both APP- 695 and the KPI-containing isoforms of APP is of primary importance, and we expect that the experiment described in this proposal will shed light on this crucial issues.
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