Differentiated P19 cells express N‐methyl‐d‐aspartate receptor 1 mRNA binding trans‐acting proteins and four N‐methyl‐D‐aspartate receptor 1 splice variants comparable to those in cultured fetal cortical neurons

Abstract

Differentiated P19 cells naturally express N-methyl-D-aspartate (NMDA) receptors and serve as a good in vitro model system with which to study NMDA receptor regulation. Here we examined expression of NR1 mRNA binding trans-acting proteins and NR1 splice variants in P19 cells. After exposure to retinoic acid, P19 cells were differentiated for 2, 4, 6, and 8 days in vitro (DIV). Total RNA and protein extracts from differentiated P19 cells were utilized to examine NR1 and NR2B expression. A steady increase in NR1 and NR2B mRNA and protein levels was observed with respect to days of differentiation. NR2B mRNA was detected within 2 DIV. However, NR2B protein appeared only at 4 DIV. By contrast, minimal expression of NR1 mRNA could be detected in undifferentiated P19 cells, whereas NR1 protein was detected at 4 DIV. RT-PCR analysis identified expression of four of eight full-length NR1 splice variants, similar to the expression pattern seen in fetal cortical neurons (FCN). These data were confirmed by ribonuclease protection assays. RNA gel shift assays and Northwestern analysis revealed the expression of NR1 mRNA binding trans-acting proteins in P19 neurons comparable to those expressed in FCN. RNA super gel shift assays confirmed the presence of the NR1 mRNA binding trans-acting protein GIIbeta in the NR1-3'UTR-P19 protein complex. Levels of GIIbeta polypeptide increased with increase in days of differentiation. Taken together, our data demonstrate that differentiated P19 cells are comparable to FCN and hence provide an excellent in vitro model for studying NR1 mRNA regulation at the posttranscriptional level.