Regulation of NMDA receptors by serine proteases tissue plasminogen activator (tPA) and plasminogen/plasmin

Abstract

Tissue plasminogen activator (tPA) is an endogenous serine protease that is found in the vascular system and the central nervous system. The tPA/plasminogen proteolytic cascade which converts plasminogen to plasmin through tPA cleavage plays a critical role in dissolving blot clots and helps to maintain vascular patency. In the central nervous system, the tPA/plasminogen system is also involved in many processes ranging from synaptic plasticity to neurodegeneration. In particular, increasing evidence implicate tPA as an important neuromodulator of the N-methyl-D-aspartate (NMDA) receptors. The aim of this thesis is to examine the modulation of NR2B-containing NMDA receptors by the tPA/plasminogen system. Through the analysis of tPA-treated rat brain lysates, I found that tPA can degrade the NR2B subunits of the NMDA receptors and this tPA-induced degradation was independent of plasmin. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment of a recombinant fusion protein containing the amino-terminal domain (ATD) of NR2B, revealed that tPA-mediated cleavage occurred at arginine 67 (Arg67) located in the ATD. Hence, I sought to examine how the deletion of a short peptide proximal to valine 68 (Val68) in the NR2B subunit, could alter NMDA receptor function. Electrophysiological studies on Xenopus laevis oocytes which heterologously expressed NR1 with the ATD-truncated form of NR2B (NR2B-&#916ATD-R67) revealed a reduction in ifenprodil sensitivity. In addition, the potencies of glycine and D-cycloserine were reduced. Furthermore, the efficacy of D-cycloserine was enhanced when the amino acids 28-67 at the proximal end of the NR2B-ATD was deleted. Although the underlying mechanisms of the findings are unknown, these findings revealed that the amino acids proximal to Val68 could harbor critical determinants that could be important for the allosteric modulation of NMDA receptor channel properties. It is unknown whether putative tPA-induced NR2B-ATD cleavage of the NR2B or other forms of modulatory mechanisms of tPA on the NMDA receptors can change the NR2B-containing NMDA receptors levels in different subcellular compartments. This paradigm was examined through the acute tPA treatment of P14 whole hippocampi and subjecting treated-hippocampi to subcellular fractionation. The subsequent analysis of the different subcellular compartments revealed that tPA treatment led to a decrease in synaptic NR2B subunit levels in the hippocampus. In addition to examining the direct modulatory role of tPA on NR2B-containing NMDA receptors, the proteolytic effect of plasmin on NMDA receptors was also investigated. Both NR1 and NR2B were found to be proteolytic substrates of plasmin. My results demonstrated that the ATD, S2 and carboxyl-terminal domain (CTD) of NR1 may harbor potential plasmin cleavage sites, which are mostly consistent with the putative cleavage sites reported by other laboratories. In addition, I found that the NR2B subunit can be cleaved by plasmin at two potential sites residing in the CTD. New insights into the modulation of NR2B-containing NMDA receptors by the tPA/plasminogen system were presented in this thesis. Further studies into the underlying mechanisms engaged by tPA in the modulation of NMDA receptors would enable us to have a better understanding of the multi-faceted roles of tPA in the brain. (500 words)