Slow egress of a mouse MHC class I molecule to the cell surface despite its strong association with β 2-microglobulin

Two H-2 D region class I genes from the wild-derived mouse strain B10.GAA37 provisionally encoding the D w16 and L w16 molecules, respectively, were transfected into mouse L cells, and the expressed gene products were analyzed serologically by flow cytometry. As expected from nucleotide sequence comparisons, these analyses revealed that several L d reactive monoclonal antibodies (mAbs) recognize L w16 and not D w16. As detected by flow cytometry of intact L.L w16 cells and B10.GAA37 splenocytes, and by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of immunoprecipitates from splenocyte lysates, the α2 domain-reactive mAb 30-5-7 detected less L w16 than did the α3 domain-reactive mAb 28-14-8, suggesting the existence of two populations of L w16 molecules: 30-5-7 + 28-14-8 + and 30-5-7 − 28-14-8 +. Sequential immunoprecipitation studies provided further evidence for these two L w16 subsets; furthermore, the 30-5-7 − 28-14-8 + subset was found predominantly on the cell surface and in association with β 2-microglobulin (β 2-m). Pulse-chase studies of B10.GAA37 splenocytes revealed that L w16, like L d, is trafficked slowly to the cell surface, whereas D w16 is trafficked quickly, like most other mouse K and D region class I molecules. Despite these similarities, L w16 and L d differ in their association with β 2-m, in that the immunoprecipitates of L w16 contained much higher levels of radiolabeled β 2-m per heavy chain. Together, these studies indicate that the slower trafficking of L w16 to the surface does not result from a weaker association with β 2-m, suggesting that other factors, such as peptide ligand-induced assembly, and/or retention by ER-resident proteins play an important role in the trafficking of major histocompatibility (MHC) class I molecules to the cell surface.