This is the first study to examine the distribution of the NMDAR, Src and MAP kinases in synaptic and extrasynaptic membranes in the developing brain and to investigate their age-related expression on the cell surface. Our major findings are: 1). At all ages, membrane-associated NMDAR and Src kinases are predominantly at synapses, whereas STEP and its substrates ERK and p38 are much more concentrated extrasynaptically. 2). There is a developmental switch from NR2B to NR2A expression in synaptic membranes with more NR1/NR2B expression in extrasynaptic membranes in the developing brain. 3). While Fyn and Src protein levels decrease with age, NR2BY1472 and NR2BY1252 mediated by these kinases are significantly higher in the adult animals. At P7, phosphorylation of NR2B at Y1336 is associated with extrasynaptic NMDAR. 4). The developmental increase in STEP is accompanied by the decrease in p38 extrasynaptically. The results imply developmental changes in expression at different locations in the coupling of NMDAR to downstream signaling mechanisms.
NR2B and Fyn are expressed at much higher levels at P7 in both synaptic and extrasynaptic membranes, suggesting the importance of Fyn in regulating NR2B in the developing brain. Fyn modulates NMDAR internalization and their lateral movement on the surfaces by phosphorylation at specific tyrosine residues. Most studies focus on NR2B Y1472, as it is the major site and the main form involved in synaptic plasticity. Little is known about the physiological function of NR2B Y1252 and NR2BY1336. From our study, although NR2B Y1472, Y1252 and Y1336 are all enriched in the synapses, NR1/NR2B is the main subunit occupying extrasynaptic sites with concomitant phosphorylation at Y1336 in the immature brain. This is in agreement with a recent study in adult hippocampal slices showing phosphorylation of Y1336 is associated with extrasynaptic enrichment of NR2B [2
]. Other studies suggested that Y1336 phosphorylation enhances calpain-mediated extrasynaptic NR2B cleavage at C terminus, which may affect the ability of NR2B binding to associated proteins and thus change downstream signaling complexes [21
]. This site also mediates activation of phosphatidylinositol 3-kinase (PI3K) and p38 dephosphorylation in mature hippocampal cultures following extrasynaptic NR2B stimulation, suggesting a possible protective role against NMDA toxicity [20
]. This phenomenon was not observed in immature cultures since NR2B Y1336 was not increased under the same condition [20
]. We found elevated NR2B Y1472, Y1252 and Y1336 expression early after neonatal HI at P7 (unpublished data), but whether and how these modifications link to their surface locations after HI and the subsequent downstream NMDAR signaling still remain unknown.
STEP61, the membrane-associated isoform, was found primarily at extrasynaptic sites in both P7 and adult brain. Correspondingly, STEP substrates ERK and p38 are mostly associated with extrasynaptic membranes. The extrasynaptic localization of STEP and p38 is consistent with a recent study from adult mouse cortical tissue [22
], it also supports the preference of p38 activation and STEP cleavage following extrasynaptic NMDAR stimulation or in vitro ischemia [22
]. STEP61 cleavage was also found in a neonatal P7 rat HI model [3
]. Compared to the adult brain, P7 animals have lower STEP and higher p38 available at extrasynaptic sites; this may be related to the greater susceptibility of neonates to HI or other brain injury involving excitotoxicity.
ERK, another STEP substrate, while more concentrated extrasynaptically, has been reported to be activated by synaptic NMDAR stimulation and shut-off by extrasynaptic NMDAR. Complex mechanisms are involved in ERK regulation, so ERK activity is determined by whether activation or inhibition dominates. The functional significance of extrasynaptic ERK and p38 is not clear. It is possible that cytosol ERK and p38 are translocated to different cell compartments including membranes to interact with specific signal proteins in response to different stimuli.
In conclusion, our study demonstrates a developmental regulation in localization and expression of NMDAR, Src and MAP kinases in synaptic and extrasynaptic membranes in mouse cortical tissue. Protein localization could contribute to, but is unlikely to fully account for the differences between synaptic versus extrasynaptic NMDAR signaling. Determining whether pro-death or pro-survival signaling following NMDAR activation predominates will allow for identification of more specific therapeutic targets for neonatal HI.