Interactions between neuroserpin and tissue plasminogen activator: possible roles in neuroserpin sorting and calcium entry

Abstract

Neuroserpin is a member of the serpin superfamily. Neuroserpin is an inhibitor of the serine protease tissue plasminogen activator (tPA) and has been shown to have roles in neuroprotection and neurite outgrowth. The most well known function of tPA is to cleave plasminogen into plasmin. tPA is expressed in the Central Nervous System and is implicated in multiple processes including synaptic regulation, synaptic plasticity and neuronal injury. Activity-dependent of tPA and neuroserpin through the regulated secretory pathway is important in these processes. Preliminary data from the Birch laboratory suggested that tPA was involved in the sorting of neuroserpin in PC12 cells to a vesicle population characteristic of the regulated secretory pathway. Recent research from other laboratories had also showed that tPA can modulate calcium levels in neuronal cells through interactions with the NMDA receptor. The goal of this research project was to further probe the importance of interactions between tPA and neuroserpin for neuroserpin sorting and tPA function. Fluorescent chimeras of tPA and neuroserpin were used to probe a possible faciltatory role for tPA in the targeting of neuroserpin to vesicles that accumulated in neurite tips, a characteristic of the regulated secretory pathway in neuronal cells. Expression of tPA-GFP led to increased accumulation of a DsRed:neuroserpin chimera in the neurite tips of NGF-treated PC12 cells. Immunocytochemistry was used to show that the sorting of endogenous neuroserpin to the neurite tips of PC12 cells was also enhanced in the presence of tPA-GFP. To elucidate the mechanism behind the sorting effect of tPA-GFP on neuroserpin, neuroserpin mutants P1 and P14 were used. The results indicated that the P1 residue which is important for the initial interaction between tPA and neuroserpin is crucial for the targeting of neuroserpin to the neurite tips by tPA. The P14 neuroserpin mutant is reactive center loop mutant of neuroserpin and proved to be important for the sorting of neuroserpin by tPA. Taken together these results indicate that interaction between tPA and neuroserpin is necessary for the effect of tPA on neuroserpin sorting.