This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.An emerging topic in current immunobiology is the vital role that glycolipid-enriched membrane rafts have in immune cell activation and functioning. Among these roles is that of antigen (Ag) presentation to T cells by antigen presenting cells (APCs). For example, a recently published study from my laboratory showed that B cell APC rafts are assembled at the site of Ag presentation, and assembly of the APC rafts was essential for efficient stimulation of the T cells. Characterization of the APC rafts also showed that: signals for raft assembly were initiated in part by MHC II, the signals were enriched in the rafts, and they resulted in actin polymerization and an actin-dependent capping of the rafts for Ag presentation. From these results, we have proposed a model where MHC II and accessory molecules that include CD48 initiate signals within membrane rafts, and this leads to actin polymerization and actin-dependent translocation of rafts to the site of Ag presentation. As MHC II is associated with the rafts, this may allow the rafts to function as a vehicle for targeting peptide bound MHC II complexes to the site of engagement with the TCR. To test our model, we will in this proposal selectively disrupt the raft-associated signaling that occurs downstream of MHC II by constitutively targeting the protein tyrosine phosphatase SHP-1 to rafts using the membrane-anchoring signal of p56lck.
Aim 1 will consist of constructing a cDNA sequence encoding the raft-targeted SHP-1 protein, as well as a second sequence encoding a SHP-1 molecule that is targeted to the non-raft fraction of cell membranes. The latter will therefore serve to control for specificity.
Aim 2 will determine the effect of the separate SHP-1 constructs on raft-associated signaling and assembly of rafts downstream of MHC II. Finally, Aim 3 will determine the effect of each construct on Ag presentation as determined by assaying activation of T cells stimulated by APCs expressing either protein. From our model, we predict that the raft-targeted SHP-1 will specifically inhibit MHC II-dependent signaling and capping of rafts, and this will coincide with a diminished ability of the APCs to stimulate T cells. Altogether, this research will instruct us regarding events leading to the onset of adaptive immune responses through presentation of Ag to T cells, and will suggest avenues that will allow us to augment or inhibit these responses.
Showing the most recent 10 out of 194 publications
