The NR1-1 C1 cassette contains a functional bi-partite nuclear localization sequence
We have demonstrated that a putative bi-partite NLS, present in the C1 cassette, is capable of mediating nuclear localization of the NR1-1 C-terminus in cultured cells and in explants of cultured rat dorsal root ganglia. Although we did not specifically test the C-terminus of NR1-3, we would expect that the NR1-3 C-terminus (C0C1C2′) would also localize in the nucleus due to the presence of the C1 cassette. A similar NLS has recently been described for a naturally occurring mutant form of human SKCa3, a Ca2+-activated K+ channel protein expressed in neurons (Miller et al. 2001). The NLS within this SKCa3 mutant imparts a very similar rapid and exclusive nuclear localization with a diffuse distribution thoughout the nucleus in transient transfection assays, as was observed for C0C1C2.
When the bi-partite NLS located in the C1 cassette was mutated, it no longer mediated active transport to the nucleus when placed in the NLS vectors used in this study. The other putative NLS in the C0 cassette do not appear capable of mediating nuclear localization of the NR1-2 or NR1-4 C-terminal cassettes in the NLS vectors. However, these putative NLS may contribute to the overall efficiency of the nuclear localization observed, as the entire C-terminal cassette of NR1-1 when placed within the NLS vector was almost exclusively located in the nucleus of transfected cells. When NR1-1 C-terminal cassettes were removed, reducing the sequence to only the C1 cassette or the NLS motif itself, the efficiency of localization to the nucleus appeared to be reduced. At present, it is not clear whether this is due to a decrease in efficiency of active nuclear import or to a reduction in nuclear retention, once transported to the nucleus. The latter may be due to the inability of these fusion proteins to interact with other nuclear proteins via the putative HLH domain, an interaction that would require the presence of both the C0 and the C1 cassettes.
Possible physiological significance of the C1 NLS
The physiological role of the NLS in the C1 cassette of NR1 remains to be elucidated. For the NR1 cytoplasmic C-terminal domain to have a physiological role, the portion of the protein that contains the NLS would have to be proteolytically liberated from the nearby transmembrane region of the NR1 subunit. Indeed, in this study we were able to detect appropriately sized peptide fragments displaying immunoreactivity against NR1 C-terminal antibodies in cells expressing NR1-1-GFP fusion proteins. Furthermore, these immunoreactive fragments were significantly enriched in nuclear extracts of 293 and PC12 cells, suggesting the potential for such cleavage and nuclear targeting to occur.
These fragments were observed regardless of the placement of GFP in the full-length NR1. Placement of GFP immediately after the fourth transmembrane region of NR1 led to the generation of cleavage products which were similar in size or slightly smaller than GFP-C0C1C2. This suggests that the proteolytic machinery that leads to the liberation of these fragments is targeted upstream or within the fourth transmembrane region, as only this type of cleavage could generate a peptide fragment that still contained C1 and C2 but was similar in size to GFP-C0C1C2. The only alternative explanation would be that the observed cleavages occurred within the GFP moiety, but if this were true, NR1-C0C1C2-GFP would not be expected to yield peptide fragments. However, peptide fragments immunoreactive for both C1 and C2 were observed.
The concept that an integral membrane protein can be cleaved, releasing an intracytoplasmic fragment that is transported to the nucleus to mediate a change in gene regulation, is not generally unique, but it is novel for the glutamate ionotropic receptors. As recently reviewed, several bacterial and mammalian transmembrane proteins are cleaved within the plasma or organelle membranes, releasing into the cytoplasm proteolytic fragments that contain NLS motifs that direct them to the nucleus and regulate gene transcription, a process now termed regulated intramembrane proteolysis or Rip (Brown et al. 2000). Notch is the best-characterized member of this family of proteins (Struhl and Adachi 1998).
Interestingly, expression of full-length Notch protein does not lead to the generation of high levels of the Notch intracellular domain (NICD). In fact, the generation of the NICD was not initially detectable (Fehon et al. 1991; Lieber et al. 1993; Rebay et al. 1993). Instead, a Gal4-VP16 transcription factor was placed within the NICD and was shown to transactivate a reporter gene, which demonstrated that the NICD was gaining access to the nucleus in response to stimulation with Delta (Struhl and Adachi 1998). Yet even these minute levels of NICD are physiologically relevant. Likewise, the amyloid precursor protein (APP), which is the other well-characterized neuronal Rip substrate, is cleaved to generate levels of the APP intracellular domain (AICD) that are barely detectable by western blot in overexpressing heterologous cells (Yu et al. 2001). The study of the NICD and AICD has been facilitated by using recombinant proteins consisting of either the intracellular domain alone, or in concert with the transmembrane region and small portions of the extracellular domain; a substrate particularly well suited for cleavage by presenilins (reviewed in Brown et al. 2000).
Along these lines, it would be surprising for Rip of NR1-1 to lead to large levels of the liberated intracellular domain. Indeed, GFP fluorescence is not observed in nuclei upon expression of NR1-C0C1C2-GFP or NR1-GFP-C0C1C2, and the C1 and C2 immunoreactive peptides are only observable upon overexpression of recombinant NR1-GFP fusion proteins. Likewise, endogenous expression of NR1 from PC12 cells did not yield detectable levels of equivalent immunoreactive peptides, which was likely exacerbated by the fact that endogenous expression of full-length NR1 was a small fraction of the expression of the recombinant fusion proteins. As with Notch, a genetic approach will likely be required to confirm or eliminate the possibility that physiologically relevant levels of the NR1 intracellular domain are generated by Rip.
The fact that not all of the NR1-1 subunit splice variants contain an NLS is not unique as the γ form of the double C2 protein contains an NLS while the membrane-associated α and β double C2 spliced variants do not, suggesting that the NLS imparts a novel although unknown function (Fukuda et al. 2001). The proteolytic cleavage of nuclear membrane-associated pro-interleukin-16 (pro-IL-16) by caspase 3 yields a protein fragment that translocates to the nucleus via a NLS sequence. The pro-IL-16 NLS protein fragment appears to function only after cleavage has occurred and nuclear accumulation is associated with growth arrest (Zhang et al. 2001). The cytoplasmic domain of neuregulin has also been shown to undergo translocation to the nucleus via a monopartite NLS in both cell lines and primary neurons (Bao et al. 1999). Neuregulins comprise a family of epidermal growth factor-related growth factors found in the central and peripheral nervous systems (Gassmann and Lemke 1997).
A search of the literature revealed that the C-terminus of the NR1 subunit is cleaved and liberated from the membrane fraction of normal murine forebrain and cerebellum in vivo (Chazot and Stephenson 1997). A cleavage of the NR1 C-terminus also appears to be induced by the extracellular protease thrombin (Gingrich et al. 2000). Thrombin is a protease that is most associated with the coagulation cascade (Goldsack et al. 1998) and it also has a well-established role in the CNS (Turgeon and Houenou 1997) and is present in high amounts after CNS trauma (Gingrich et al. 2000). The observed cleavage appeared to liberate a 12-kDa fragment of the NR1-1 C-terminus, which corresponds to the predicted weight of the intact C0, C1, and C2 cassettes. This would suggest that thrombin was directing cleavage to the juxtamembrane region of the extracellular fourth transmembrane domain of NR1-1. However, the authors of this study were unable to conclude that cleavage occurred within the intracellular space (Gingrich et al. 2000). Since there is no consensus thrombin cleavage site (according to the scheme of Backes et al. 2000) in either the intracellular, transmembrane or extracellular juxtamembrane region of NR1, thrombin may not be acting directly on NR1-1. Also, if the observed cleavage were truly intracellular, then thrombin must transmit an activating signal through the plasma membrane to direct the cleavage of NR1-1. Although there are several G protein-coupled receptors for thrombin (Coughlin 1999) that could possibly mediate such a signal, Gingrich et al. (2000) demonstrated convincingly that the observed thrombin-mediated cleavage of NR1-1 was independent of the best-characterized thrombin receptor, the protease activated receptor-1 (PAR1).
Interestingly, mice that overexpress the thrombin inhibitor protease nexin-1 (PN-1) exhibit enhanced NMDAR synaptic transmission, and PN-1 knockout mice manifest a reduction in NMDAR transmission that might be associated with reductions in the number of functional receptors (Luthi et al. 1997). As well, presenilin-1 knockout mice are more susceptible to glutamate excitotoxicity (Guo et al. 1999) and glutamate excitotoxicity appears to play a prominent role in Alzheimer disease pathogenesis (Guo et al. 1999). All of these data are consistent with a role for cellular proteases in directing Rip of NR1-1, although other explanations for these results are possible. Tissue plasminogen activator has also been recently demonstrated to interact with NR1-1, resulting in its cleavage and potentiation of NMDA receptor-mediated excitotoxicity (Nicole et al. 2000).
The physiological role of the NR1-1 C-terminal fragment as well as many of the newly described NLS motifs in membrane-associated proteins and proteolytic fragments remains to be determined (Healy et al. 1999; Craggs and Kellie 2001; Fukuda et al. 2001; Miller et al. 2001). The NR1-1 C1 cassette NLS may regulate expression and/or transcription of NR1 and NR2 or affect the regulation of expression of other genes. The C-terminus contains regions that resemble the HLH secondary structure of the myc family of transcription factors. It is interesting to note that one of the SREBP proteins (human SREBP2), a known member of the Rip family of proteins, also has a myc-type HLH domain, homologous to that of the putative HLH domain present in the C0C1 region of NR1-1, and appears to be involved in regulating gene transcription (Hua et al. 1993). This also raises the possibility that the cleavage of the NR1-1 C-terminus may occur at intracytoplasmic membranes. An endoplasmic reticulum retention sequence has recently been demonstrated to exist within the C1 cassette (Standley et al. 2000). Future experiments will be required to determine the significance of the NLS and the putative myc-type HLH protein–protein structural motif in the cytoplasmic C-terminal domain of NR1-1. Two calmodulin binding domains span these same C0C1 regions (Ehlers et al. 1996) and NMDA receptor activation induces translocation of calmodulin to the nucleus in a facilitated manner where it interacts with CaMKIV to alter the phosphorylation of the CREB transcription factor (Deisseroth et al. 1998). The binding of calmodulin to the NR1-1 C-terminus, and subsequent cleavage, may represent a potential mechanism by which calmodulin is targeted to the nucleus through interaction with a released C0C1 domain. A proteolyticly released NR1-1 C-terminal fragment may have a role in the development of the nervous system and in known NMDAR-associated functions such as learning and memory. Alternatively, the proteolytic cleavage of NR1-1, released as a consequence of the presence of abnormal levels of proteases such as thrombin after a CNS trauma, may yield proteolytic fragments with dominant negative effects that could contribute to glutamate-mediated excitotoxicity. We have already demonstrated that the expression of the NR1-1 C-terminus in DRG neurons results in a dominant negative effect that blocks membrane placement of NR1-1 subunits and, in a significant proportion of DRG neurons, induced apoptotic-like nuclear fragmentation (Marsh et al. 2001). This dominant negative effect is similar to that recently described for the mutant form of human SKCa3 (Miller et al. 2001). Together these results suggest that, under the appropriate circumstances, a released NR1-1 C-terminal peptide could have important physiological consequences.