Truncated MHC class II cytoplasmic and transmembrane domains: Effect on plasma membrane expression

• Published in: Molecular immunology 1995-04, Vol.32 (6), p.433-446
• Main Authors: Wade, William F., Khrebtukova, Irina, Schreiber, Kathy L., McKean, David J., Wade, Terri K.
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Differentiation, B-Lymphocyte; Base Sequence; Biological Transport; Cell Compartmentation; Cell Membrane - metabolism; Cytoplasm - chemistry; cytoplasmic domains; DNA Primers - chemistry; Gene Expression; Hexosaminidases - pharmacology; Histocompatibility Antigens Class II - chemistry; Histocompatibility Antigens Class II - genetics; Histocompatibility Antigens Class II - metabolism; Immunologic Techniques; In Vitro Techniques; invariant chain; MHC class II; Mice; Molecular Sequence Data; plasma membrane expression; Protein Conformation; RNA, Messenger - genetics; Structure-Activity Relationship; Transfection; transmembrane
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/0161-5890(94)00165-W

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IA k's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-A k molecules. A kβTM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-A k conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the A kβ chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the A kβ chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to access a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the A kαCy domains is proposed to