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 been studied using both transiently and permanently transfected mammalian cell lines. The expression of either NR1a or NR2A alone does not result in significant cell surface expression of either subunit as determined by cell surface biotinylation and immunofluorescence staining. When NR1a is expressed alone large intracellular accumulations of the subunit are formed which do not co-localize with the golgi apparatus markers protein p58 and wheat germ agglutinin, but do co-localize with the endoplasmic reticulum marker calreticulin. Co-expression of NR1a and NR2A results in a reduction of these intracellular accumulations and the appearance of both subunits on the cell surface. Immunoprecipitation 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 [35S]methionine in the presence of Brefeldin-A, a drug which prevents protein transport from the endoplasmic reticulum, NR2A could be immunoprecipitated by an antiserum specific for NR1a. Together these results suggest that the NMDA receptor subunits are assembled in the endoplasmic reticulum and that co-synthesis of the subunits is necessary for their association and their successful cell surface targeting.
McIlhinney
R A
RA
Medical Research Council, Anatomical Neuropharmacology Unit, Oxford, UK.
Le Bourdellès
B
B
Molnár
E
E
Tricaud
N
N
Streit
P
P
Whiting
P J
PJ
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D011233:Precipitin Tests; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D014162:Transfection
D016194:Receptors, N-Methyl-D-Aspartate