Post-translational regulation of the NKG2D ligand Mult1 in response to cell stress

NKG2D is a major stimulatory receptor expressed by natural killer (NK) cells and some T cells. The receptor recognizes MHC class I-like cell surface ligands that are poorly expressed by normal tissues but often induced in transformed and infected cells. The existence of several NKG2D ligands in each individual, some with strikingly divergent protein sequences, raises the possibility that different ligands are regulated by distinct disease-associated stresses. The transcripts for some ligands, including murine UL16-binding protein-like transcript 1 (Mult1), are abundant in certain normal tissues where cell surface expression is absent suggesting the existence of translational or post-translational regulation.;This dissertation establishes that Mult1 is ubiquitinated on lysine residues within its cytoplasmic tail, targeting the protein for degradation. Inhibition of lysosomal acidification leads to accumulation of Mult1 at the cell surface and within lysosomes indicating that they are the primary site for Mult1 degradation. This pathway may explain the lack of inappropriate Mult1 expression in healthy tissues where high levels of Mult1 transcripts are present.;Mult1 degradation and ubiquitination is reduced in response to stress imparted by ultraviolet (UV) irradiation. Other genotoxic agents do not induce Mult1 at the protein level, indicating that this pathway is distinct from the previously identified regulation of NKG2D ligands by genotoxic stress. The heat shock pathway also induces Mult1 at the protein level, suggesting that Mult1 may be specifically induced by protein folding stresses.;Membrane-associated RING-CH (MARCH)-4 and MARCH-9 are capable of regulating Mult1 expression in response to heat shock, but may overlap with other E3 ligases in this regulatory role. The MARCH family of E3 ligases appears to have an important role in controlling expression of a broad range of transmembrane proteins with relevance to the immune system. The finding that MARCH-9 is involved in Mult1 regulation extends the immuno-regulatory role of these E3 ligases to cover NKG2D ligands.;These findings represent a novel mechanism for stress-mediated cellular control of NKG2D ligand expression. There is now evidence for regulation of NKG2D ligands at multiple stages of gene expression, suggesting that integration of multiple pathways is required for activation of lymphocytes through NKG2D. Protein degradation is a final layer of Mult1 regulation, only allowing Mult1 to accumulate at the cell surface if certain stresses are present. It will be of interest in the future to determine which cells and pathological conditions utilize this regulatory mechanism to activate the immune system.