Turnover analysis of N-methyl-D-aspartate receptor subunit NR1 protein in PC12 cells

The post-translational fate of N-methyl-D-aspartate receptor (NMDAR) subunit NR1 was characterized in PC12 cells using pulse-chase labeling, block of protein synthesis by cyclohexamide and deglycosylation by endoglycosidase H. Metabolic labeling of NR1 protein indicated a biphasic degradation of NR1 protein with half-lives of 1.6 and 16.1 h for a rapidly (78%) and a slowly (22%) degrading population. Immunoprecipitation of NR1 following the block of protein synthesis by cyclohexamide revealed that the rapidly and slowly degrading pools mainly consisted of the NR1 splice variants NR1-4a and NR1-2a. Sensitivity of NR1 protein to deglycosylation by endoglycosidase H indicated the presence of an immature form of NR1 that was retained in the endoplasmic reticulum. PC12 cells serve as a useful model for the elucidation of translational and post-translational mechanisms of NMDAR expression.