The overall goal of this project is to use CD73 as a model system to investigate how glycosyl phosphatidylinositol (GPI)-anchored proteins, which lack cytoplasmic domains, participate in signal transduction.Major advantages of studying CD73 are the variety of experimental conditions in which CD73 mAbs are mitogenic, the magnitude of the T cell response to CD73 mAbs as compared to mAbs to other GPI-anchored proteins, and the availability of panels of CD73 mAbs and the applicant's own transfected cell lines expressing GPI-anchored and mutant forms of CD73. The applicant will also attempt to demonstrate a physiologic role for CD73 in T cell activation by identifying its natural ligand.
The specific aims are: 1. To delineate the mechanism by which human T cells are activated by mAbs to GPI-anchored cell surface proteins. Jurkat transfectants expressing high levels of GPI-anchored CD73 will be stimulated with CD73 mAbs and examined for Ca+2 mobilization, PIP2 turnover, and protein tyrosine phosphorylation. The importance of CD3/TCR and pp56lck to signalling via GPI- anchored molecules will be determined with Jurkat mutants defective in CD3/TCR or pp56lck and transfected with CD73. The role of the GPI anchor will be determined by studying Jurkat transfectants expressing CD73 with a conventional transmembrane domain. Multiple approaches will be exploited to determine whether CD73 and other GPI-anchored molecules are physically associated with known (or novel) signal transducing proteins. 2. To identify and characterize a natural ligand for CD73 using a soluble chimeric CD73Ig fusion protein as a probe. 3. To determine the mechanism by which CD73 expression is down-regulated after interaction with immobilized CD73 mAb. It is anticipated that the results of this investigation will give new insight into the function of CD73 in the human immune system and the mechanism by which it and other GPI-anchored proteins transmit activation signals to T cells.
