The role of transmembrane domains in the March-I-mediated downregulation of MHC-II and CD86.

Abstract MHC class II molecules on APCs bind to and presents peptides to antigen-specific CD4 T cells. DCs are the most potent subset of APC and can activate naïve T cells. Immature DCs possess relatively low expression of the costimulatory molecule CD86 and have a rapid rate of turnover of MHC-II. This is due in part to ubiquitin-mediated degradation of MHC-II and CD86 by the transmembrane (TM) ubiquitin ligase March-I in immature DCs. Curiously, March-I activity is suppressed by CD83, a TM protein that is upregulated by DC activation. Despite the well-documented role of March-I in specifically regulating MHC-II and CD86 expression, little is known about how these substrates interact with March-I and how CD83 is able to suppress March-I function. In this study we have examined the importance of TM domain interactions in March-I function by replacing TM domain sequences in March-I or CD83 with that of ICAM-1. A March-I TM1 mutant had reduced ability to affect MHC-II expression and had no effect on CD86 expression. Binding studies demonstrated that that March-I TM1 mutant lost all ability to bind to CD86. Whereas wild-type CD83 was able to block March-I downregulation of MHC-II and CD86, replacement of the CD83 TM domain with that of ICAM-1 completely abrogated CD83 suppression of March-I function. Binding studies confirmed that the CD83 TM mutant had lost all ability to bind to March-I. Our data therefore suggest that March-I binds to MHC-II and CD86 by TM domain interactions and that CD83 blocks March-I function by competitively inhibiting the binding of March-I to MHC-II and CD86.