The specific objectives of this proposed study are: (1) to investigate the hypothesis that the T-lymphocyte transmembrane glycoprotein CD45 (GP180), closely interacts with the T cell antigen receptor/CD3 complex (TCR/CD3 complex); and to analyze the linkage between CD45(GP180) and the cytoskeleton in the T-lymphocyte plasma membrane; and (2) to identify the IP3 receptor on Ca2+ storage vesicles (calciosomes) responsible for IP3- induced internal CA2+ release during lymphocyte activation. The employment of complementary techniques including density perturbation technique [for CD45(GP180) patch/cap isolation], non-ionic detergent extraction (e.g. Triton X-114) and Con A-Sepharose column chromatography should allow us to isolate the putative CD45 (GP180)-TCR/CD3 complex and identify substrates of the CD45 (GP180)-tyrosine phosphatase in these receptor complexes as well as the relevant CD45(GP180)-cytoskeleton complexes. The proposed experiments concerning reconstitution of CD45(GP180) into liposomes (phospholipid vesicles) will allow us to directly examine any direct interactions which occur between CD45(GP180) and various cytoskeletal components. Furthermore, the experiments proposed to test the effect of the cytoskeleton on CD45(GP180)-associated tyrosine phosphatase activity should provide new insights concerning the regulatory role of the cytoskeleton in signal transduction. In addition, we propose to identify the IP3 receptor on Ca2+ storage vesicles (calciosomes) responsible for IP3-induced internal Ca2+ release during lymphocyte activation. A variety of methods including cellular fractionation, density gradient centrifugation, radiolabel binding assays, CA2+ flux measurements and immunocytochemistry, will be used to analyze both the IP3 receptor and IP3-induced internal Ca2+ release mechanism which is required for the onset of lymphocyte activation by a specific ligands (e.g. anti-TCR or anti-CD3 antibodies). We believe that the proposed experiments will allow us to obtain valuable new insight(s) concerning (a) the relationships between CD45 (GP180) and TCR/CD3 complex during T-cell activation; (b) the functional role of the cytoskeleton in regulating signal transducing systems (e.g. tyrosine phosphatase activity); and (c) the molecular and cellular mechanisms involved in regulating internal Ca2+ release from intracellular storage sites. This knowledge is critically needed to further our understanding of lymphocyte activation during various immunological reactions and certain immunodeficiency-related diseases.
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