Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor subunit, NR1, in cultured cerebellar granule cells

The Plumber, composition, and location of receptors in neurons are critically important factors in determining the neuron's response to neurotransmitters. The functional expression of receptors appears to be regulated both generally, at the level of transcription or translation, and locally, at the level of the individual synapse, A key component in the regulation of any protein is its turnover rate, which, measured in half-lives, ranges from a few minutes to several days. In the present study, we measured the turnover rates of subunits of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, the two major ionotropic glutamate receptors, using cultured cerebellar granule cells. Turnover rates for NR1, NR2A/B, GluR2/3, and GluR4 subunits were determined by pulse-chase labeling of cells with [S-35]methionine. Half-lives were found to be 18 +/- 5 h and 23 +/- 8 h for the AMPA receptor subunits GluR2/3 and GluR4, respectively, and 16 +/- 5 h for NR2A. The NR1 subunit showed a biphasic decay with half-lives of 2 and 34 h for the rapidly and slowly degraded populations, respectively, Splice variants of the NR1 subunit with different carboxyl-terminal cassettes, C2 and C2', showed similar biphasic degradation patterns, To further characterize the rapidly degraded pool of NR1, surface receptors were labeled by biotinylation, and half-lives of the biotinylated proteins were determined, All surface NR1 was slowly degraded with a pattern similar to that of NR2A, GluR2/3, and GluR4, suggesting that the rapidly degraded pool is confined to the cytoplasm and not assembled with NR2 subunits, A significant amount of NR1 was not immunoprecipitated by NR2 subunit-specific antibodies after solubilization with deoxycholate. This unassembled pool, but not the assembled one, was greatly diminished following treatment of cycloheximide for 5 h, indicating that the rapidly degraded pool of NR1 is not assembled with NR2, These results show that MMDA and AMPA receptors have similar turnover rates, but NMDA receptors have a separate pool of NR1 subunits that is rapidly degraded and accounts for most of the intracellular pool.