Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.

N.J Sucher,N Brose,D.L Deitcher,M Awobuluyi,G.P Gasic,H Bading,C.L Cepko,M.E Greenberg,R Jahn,S.F Heinemann,S.A Lipton

doi:10.1016/s0021-9258(18)41528-1

Abstract

Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.

Highlights

A'-methyl-D-aspartate receptor was detectebdy North- receptor is involved in fast excitatory synaptic transmission ern hybridization in both nerve growth factor-differ- and neuronal plasticity in the centralnervous system (CNS), entiated and undifferentiated rat pheochromocytoma and its over-stimulation cacnause neuronal degeneration and (PC12) cells
Northern blot analysis revealed that messenger RNA for the NMDARl subunit of the NMDARl subuniotf the The I?-methyl-D-aspartate (NMDA) receptor is abundantly expressed in PC12 cells
No functional NMDA-operated channels were found in PC12 cells and immunodetection with a monoclonal antibody indicated the presence of only trace amounts of NMDARl protein in this cell line

Summary

EXPERIMENTAL PROCEDURES

Cell Cultures-PC12, human embryonic kidney 293 cells, hippocampal, and astrocyte cultures were prepared as described previously (McCarthyand De Vellis,1980; Greenberg and Ziff, 1984; Bading and Greenberg, 1991). The NMDA probe template was a -1.7-kb cDNA fragment corresponding to NMDARl that was obtained by polymerase chain reaction (PCR) from a hippocampal cDNA expression library and subcloned into a plasmid (Bluescript KS(+), Stratagene). Protein amounts in each sample were determined with the Micro BCA protein assay reagent kit from Pierce Chemical Co. Transfection of PC12 Cells and the Human Embryonic Kidney Cell Line 293-The NMDARl clone pJSl (isolated from a rat forebrain library and cloned into Bluescript SK(-)) or the BamHI/EcoRV fragment of clone pBKS/505 (see above)were inserted into the mammalian expression vector pcDNAI/AMP (Invitrogen). The bath solution contained Hanks' physiological saline with 2.5 mM CaClzplus 1p~ glycine (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988); MgZf was omitted to prevent its voltagedependent block of NMDA receptor-operated channels (Mayer et al, 1984; Nowaket al., 1984).The cultures were continuously superfused (-0.8 ml/min) inthis solution a t room temperature. Agonists (NMDA-glycine, glutamate, dimethylphenylpiperazinium (DMPP), or high K+)were applied by superfusion. [Ca2+]w, as measured 20 s after addition of the agonist

RESULTS

Findings

DISCUSSION