ASSEMBLY INTRACELLULAR TARGETING AND CELL-SURFACE EXPRESSION OF THE HUMAN N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS NR1A AND NR2A IN TRANSFECTED CELLS

The intracellular trafficking, assembly, and cell surface targeting of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A has be en studied using both transiently and permanently transfected mammalia n cell lines. The expression of either NR1a or NR2A alone does not res ult in significant cell surface expression of either subunit as determ ined by cell surface biotinylation and immunofluorescence staining. Wh en NR1a is expressed alone large intracellular accumulations of the su bunit are formed which do not co-localize with the golgi apparatus mar kers protein p58 and wheat germ agglutinin, but do co-localize with th e endoplasmic reticulum marker calreticulin. Go-expression of NR1a and NR2A results in a reduction of these intracellular accumulations and the appearance of both subunits on the cell surface. Immunoprecipitati on of NR1a from in vitro translated subunit proteins showed that NR2A could only be immunoprecipitated with NR1a when both subunits were co- synthesized in the presence of microsomes. When cells expressing NR1a and NR2A were incubated with [S-35]methionine in the presence of Brefe ldin-A, a drug which prevents protein transport from the endoplasmic r eticulum, NR2A could be immunoprecipitated by an antiserum specific fo r NR1a. Together these results suggest that the NMDA receptor subunits are assembled in the endoplasmic reticulum and that co-synthesis of t he subunits is necessary for their association and their successful ce ll surface targeting. (C) 1998 Elsevier Science Ltd. All rights reserv ed.