Ecto-5' -nucleotidase (ecto-5' -nt) is a differentiation antigen expressed on subpopulations of human T and B cells. The majority of ecto-5' -NT activity can be released from the lymphoycte surface by phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that at least a portion of this enzyme is attached to the membrane by covalent linkage to PI or a closely-related structure. Recent experiments in our laboratory suggest that ecto-5' -NT, like other PI-linked T cell surface antigens including Thy-1, T cell activating protein, and Rt-6, may be involved in transmembrane signaling, since anti-5' -NT antibodies, in combination with submitogenic doses of phorbol esters, can drive human T cells to proliferate. The purpose of the proposed research is threefold: 1) to understand how the expression of ecto-5' -NT is regulated during human lymphocyte ontogeny and differentiation, 2) to delineate the molecular mechanism(s) responsible for reduced ecto-5' -NT expression in lymphocytes of patients with immunodeficiency diseases, and 3) to determine whether the PI-containing membrane anchoring structure of ecto-5' -NT is necessary for its proposed function in transmembrane signaling. Therefore, we plan to clone the gene for 5' -NT in order to develop probes which can be used to study the transcriptional control of ecto-5 -NT expression and the structure of the 5' -NT gene, including regulatory sequences. 5' -NT specific mRNA from ecto-5' -NT positive and negative T and B cells from normal individuals, as well as from lymphocytes of immunodeficient patients with ecto- 5 -NT deficiency, will be characterized by Northern blotting. We will attempt to identify cDNAs corresponding to a PI-linked form of the enzyme, an integral membrane form, and perhaps a soluble form. We will then determine whether ecto-5 -NT deficiency results from a failure to transcribe the 5'-NT gene or Whether a form of the enzyme is synthesized which cannot be inserted into the membrane. We will also transfect the ecto-5 -NT- human T cell line Jurkat with 5'-NT cDNAs corresponding to PI-linked and integral membrane forms of 5'-NT. In order to determine whether the PI anchor of 5' -NT is necessary for the enzyme to function in signal transduction, both types of transfectants will be tested for the ability to secrete IL-2 after stimulation with anti-5 -NT antibodies and phorbol esters. The results of these experiments will not only give insight into the mechanisms controlling ecto-5'-NT expression during lymphocyte differentiation, but will also increase our understanding of the function of this interesting enzyme and the significance of reduced ecto-5 -NT expression on lymphocytes of immunodeficient patients.
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