TRUNCATED MHC CLASS-II CYTOPLASMIC AND TRANSMEMBRANE DOMAINS - EFFECTON PLASMA-MEMBRANE EXPRESSION

Plasma membrane (PM) expression of major histocompatibility complex (M HC) 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 transmem brane (TM) and cytoplasmic (Cy) domains, in an attempt to understand w hat their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IA(k)'s alpha of beta chain, or both, we have identified some of the sequence requirements for effi cient surface expression of I-A(k) molecules. A(k) beta TM mutants tha t are not expressed at the PM are not transported past the medial-Golg i as indicated by in situ staining and Western blot analysis of endogl ycosidase-H-treated immunoprecipitates. The lack of transport of TM cl ass II mutants is not due to lack of association with the invariant ch ain (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 r evealed a discrete vesicular pattern compared to the staining of trans fectants that expressed wildtype class II molecules. The immunofluores cence data along with the endoglycosidase-H data indicate the Cy domai ns are required for efficient transport. Immunoprecipitation studies u sing a panel of I-A(k) conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the con formation of class II. However, further truncation of the A(k) beta ch ain into the TM domain resulted in lack of transport past the ER/media l-Golgi and diminished expression (stability) of mutant class II prote ins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except f or one, 3F12. We conclude that the Cy domain of the alpha and beta cha ins of MHC class II, as well as sequences in the TM domains of the A(k ) beta chain are required for efficient class II PM expression. The re ason for the lack of PM expression of TM mutants may be the inability to access a transport competent conformation as defined by the 3F12-sp ecific epitope, while truncation of the A(k) alpha Cy domains is propo sed to prevent complete masking of the ER retention sequence of the Ii chain.