Abstract. Class I major histocompatibility (MHC) antigens are expressed by virtually all mammalian cells, yet their levels of expression and behavior on the cell surface vary in a cell-specific fashion. A panel of lymphoid (both B and T) and nonlymphoid cell lines was used to study the kinetics of internalization of the H-2L d class I MHC in different cell types. These studies revealed that endocytosis of H-2L J occurs by both constitutive and PMA-regulated pathways in lymphoid cells, but only by a PMA-refractory pathway in the nonlymphoid cells tested. Transfectant derivatives of the T lymphoma, EL4, which express wild-type or mutant H-2L d class I MHC antigens, were used to investigate the requirement for the cytoplasmic domain of the class I MHC antigen for its endocytosis in T lymphocytes. These studies showed that modification or deletion of the cytoplasmic domain of H-2L ~ abrogates endocytosis via a PMA-regulated pathway. The role of cytoplasmic domain phosphorylation in PMAinducible endocytosis was examined. The wild-type H-2L d antigen is phosphorylated in all cell types examined, and this phosphorylation is up-regulated by PMA treatment. In contrast, cytoplasmic domain mutants of H-2L d fail to be phosphorylated in vivo, in the presence or absence of PMA. The universality of PMA-inducible hyperphosphorylation of the class I MHC antigen among diverse cell types leads us to conclude that phosphorylation of the cytoplasmic domain, while perhaps necessary, is not sufficient for triggering endocytosis via a PMA-inducible pathway. Furthermore, the results with the cytoplasmic domain mutants of H-2L ° suggest that a structural conformation of the class I MHC cytoplasmic domain is required for endocytosis via this route.

Directed and Brownian movement of class I major histocompatibility complex (MHC) molecules on cell membranes is implicated in antigen presentation. Previous studies indicated that the class I MHC cytoplasmic tail imposes constraints on the molecule's diffusion. Here we used single particle tracking to study the mobility of the wild-type mouse H-2L(d) class I MHC molecule and of seven cytoplasmic tail variants. Six of the variants have cytoplasmic tails of four or seven residues (differing in net charge), and one is tailless, yet all are susceptible to confinement in membrane domains. However, truncation of the cytoplasmic tail to 0-4 residues decreases the proportion of particles exhibiting confined diffusion and increases the proportion exhibiting simple diffusion. Particularly for the truncated mutants (tail length of 0-7 residues), many of the particles have complex trajectories and do not move at a constant speed or in the same mode of diffusion throughout the observation period. Several particles of the tailless H-2L(d) mutant display a type of directed diffusion that is rarely observed for other H-2L(d) mutants. Taken together, these data show that even short cytoplasmic tails can influence markedly class I MHC mobility and that cytoplasmic tail length and sequence affect the molecule's diffusion in the membrane.

No abstract

Class I MHC molecules have been thought to occur in vivo both as class I MHC heavy chain-beta 2-m heterodimers, which are or are not associated with antigenic peptide, and as free class I MHC heavy chains. Class I MHC molecules are now found also to occur in another type of structure: a heavy chain-heavy chain dimer. Biochemical studies show that heavy chain dimers are disulfide-linked via a conserved cytoplasmic domain cysteine. H-2Ld, H-2Db, and H-2Dd class I dimers fail to react with certain alpha 1 and alpha 2 domain-specific antibodies. Furthermore, although beta 2-m-specific antibodies coprecipitate class I MHC heavy chains, they do not coprecipitate class I MHC heavy chain dimers. Pulse-chase studies show that heavy chain dimer formation occurs at different points in the biosynthesis of class I MHC molecules in beta 2-m+ and beta 2-m- cells: in beta 2-m+ cells, heavy chain dimers form after the class I molecules have traversed the medial Golgi cisternae, whereas in beta 2-m- cells they form immediately. Culturing of beta 2-m+ cells with exogenous beta 2-m prevents the formation of H-2Ld/Db heavy chain dimers. We conclude that dimer formation occurs as a consequence of loss or unavailability of beta 2-m. Class I MHC heavy chain dimerization may provide a mechanism for removal of immunologically dysfunctional molecules.

No abstract