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

Abstract

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.