A POLYMORPHIC RESIDUE IN THE AMINO-TERMINAL ALPHA-1 HEMI-DOMAIN OF THE MOUSE L(D) CLASS-I MOLECULE AFFECTS ITS ASSEMBLY AND SURFACE EXPRESSION

In comparison to D-d and most other mouse major histocompatibility com plex class I molecules, the L(d) molecule is poorly expressed on the c ell surface, has a lower affinity for beta(2)-microglobulin and is tra fficked more slowly to the cell surface. Previous studies using L(d)-D -d exon-shuffled constructs and the chimeric D-dml molecules suggested that the L(d) alpha 1 domain was responsible for this phenotype. Two constructs, one containing an L(d)-D-d hemi-exon-shuffled alpha 1 exon and the other containing a D-d-L(d) hemi-exon-shuffled alpha 1 exon, were inserted into either L(d) or D-d to replace the intact alpha 1 ex on. These constructs were transfected into mouse L cells. Flow cytomet ric analyses of the resulting transfectants indicate that the D-d-L(d) alpha 1/L(d) molecules, similar to the D-d alpha 1/D-d alpha 2/L(d) m olecules, were expressed at a higher level on the cell surface than ei ther the L(d)-D-d alpha 1/L(d) molecules or intact L(d) molecules. Ana lyses of the molecules in lysates suggested that a higher proportion o f the D-d-L(d) alpha 1/L(d) molecules, like the D-d alpha 1/D-d alpha 2/L(d) molecules, as compared to the L(d)-D-d alpha 1/L(d) and intact L(d) molecules were assembled as detected by alpha 2 domain-reactive m onoclonal antibodies. Pulse-chase and lysate stability studies suggest ed that the lower steady state levels of assembled L(d)-D-d alpha 1 mo lecules resulted from a slower assembly rate rather than instability. Collectively, these studies suggest that residues in the amino termina l half of the L(d) alpha 1 domain are responsible for its inefficient assembly, probably leading to its low cell surface expression. To dete rmine which polymorphic residues in the amino terminal alpha 1 hemi-do main might influence this phenotype, several L(d) point mutants, in wh ich a D-d amino terminal alpha 1 hemi-domain residue was substituted i nto the corresponding position of L(d), were analysed. These analyses suggested that, while the residue at position 9 has only a slight effe ct on beta(2)-microglobulin association, it has a striking effect on a ssembly and cell surface expression.