The murine major histocompatibility complex (MHC) class I cell-surface glycoproteins H-2 and Qa-2 are greater than 80% homologous in sequence, but have distinct biological functions. Classical H-2 transplantation antigens are transmembrane proteins present on all somatic cells that bind numerous peptides derived from intracellular degraded protein antigens and present these to CD8+ effector T cells. In contrast to H- 2, Qa-2 is covalently linked to a glycosyl phosphatidylinositol (GPI) anchor that does not span the lipid bilayer. In general, the signals that mediate on mature T cells and apparently does not function in antigen presentation. A logical reason for this functional difference is that the type of membrane anchor influences trafficking of the molecule through the cell. A number of studies have shown that GPI- anchored proteins can activate cells to proliferate, but it is unclear whether this is a direct property of the anchor itself, the interaction of these proteins with other signal transducing molecules, or both. Therefore, using Qa-2/H-2 as a model system, this proposal will investigate the nature of GPI-attachment signals, the effect of GPI anchors on MHC-I function, the role of GPI-anchored molecules in cell activation, and the differences in intracellular trafficking between GPI-anchored and transmembrane MHC-I. GPI anchor-attachment signals in the Qa-2 protein Q7b will be characterized using exon exchange and site-directed mutagenesis, followed by expression of mutant proteins in well characterized R1.1 cells. Accomplishment of these objectives should reveal more about the nature of GPI attachment signals in general, and generate a number of constructs that express wild type and mutant proteins useful for studies of MHC-I function and trafficking. Cell lines will be transfected with constructs encoding isoforms of MHC-I that differ in their mode of anchorage in order to test their ability to present antigen. These studies should provide a better understanding of the functional consequences of linkage to GPI, and the basis for biological differences between H-2 and Qa-2. The ability of GPI-anchored versus transmembrane forms of Q7b and Db to activate T cells will be determined by transfection of a T cell clone that can be assayed for proliferation and IL-4 gene activation, to test the hypothesis that the GPI anchor itself is directly involved in the activation process. In cells that demonstrate functional differences between GPI-anchored and transmembrane forms of Q7b and Db, differences in intracellular trafficking will be investigated biochemically and by fluorescence and electron microscopy. The effects of various inhibitors of vesicular transport will be examined to test the hypothesis that GPI-anchored proteins are transported through different exocytic and endocytic pathways. These studies should provide a better understanding of the relationship between MHC-I protein trafficking and function, with regard to antigen processing and signal transduction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM046467-05
Application #
2183956
Study Section
Immunobiology Study Section (IMB)
Project Start
1991-08-01
Project End
1996-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Kurian, M S; de la Cuesta, R S; Waneck, G L et al. (1995) Cleavage of Mullerian inhibiting substance activates antiproliferative effects in vivo. Clin Cancer Res 1:343-9
Margolese, L; Waneck, G L; Suzuki, C K et al. (1993) Identification of the region on the class I histocompatibility molecule that interacts with the molecular chaperone, p88 (calnexin, IP90). J Biol Chem 268:17959-66
Kamitani, T; Chang, H M; Rollins, C et al. (1993) Correction of the class H defect in glycosylphosphatidylinositol anchor biosynthesis in Ltk- cells by a human cDNA clone. J Biol Chem 268:20733-6
Cherayil, B J; MacDonald, K; Waneck, G L et al. (1993) Surface transport and internalization of the membrane IgM H chain in the absence of the Mb-1 and B29 proteins. J Immunol 151:11-9