The study of the initiation and regulation of growth factor-mediated signal transduction pathways is of crucial importance to the eventual understanding of the mechanisms controlling cell growth and proliferation. The loss of control of this apparatus is thought to be a hallmark of malignant transformation and autoimmune diseases. This laboratory has focussed on T lymphocytes as a model system to study the first biochemical steps in the activation of resting cells. We had previously shown that stimulation of the antigen receptor complex rapidly lead to an increase in cellular Ca2+ and phosphatidylinositol turnover. During the course of these studies we discovered that the exotoxin of Bordetella pertussis, """"""""PTx"""""""", was a very potent mitogen for human T cells. We have shown that the toxin specifically binds to a previously unknown 43 kDa plasma membrane protein in human T lymphocytes; this receptor is responsible for generating the mitogenic signal. We have recently found that p43 is also expressed on B cells and monocytes. The 43 kDa PTx binding protein/receptor is associated with a 58 kDa protein the expression of which is also not limited to T cells, as it is present on neutrophils, B cells, monocytes, and all thymocytes. Mutant cell lines lacking p58 are defective in both PTx and antigen receptor-stimulated signal generation. A monoclonal antibody against p58 is by itself moderately mitogenic for T cells, and is synergistic when used with PTx, phytohemagglutinin, or mitogenic anti-T cell receptor antibodies. We have also isolated an anti-p43 antibody. There is evidence to suggest that this complex functions as cell adhesion molecules. The purpose of this application is to continue our studies of this receptor complex. We will focus on two aspects of this system: continuation of the structural and functional studies and obtaining cDNA clones for the two proteins. We will continue to characterize and isolate mutant cell lines which are p43(-), p58(-), and p43/p58(-). We will investigate the nature of the physical association between p43 and p58 by further crosslinking and co-immunoprecipitation studies. Since p58(-) mutants appear to be defective in antigen receptor-mediated signalling, we will investigate the interaction of p58 and the T cell receptor. We will continue to study the ontogeny of p58 and p43 expression in both T cells/thymocytes and the other hematopoietic cells on which the former is expressed; this will be done by flow cytometry and immunoprecipitation. We will study the mechanism by which the anti-p58 monoclonal antibody is a co- mitogen for T cells. We will study the ability of anti-p58 and anti-p43 antibodies to block cell-cell interactions in the activation of T cells. W will isolate cDNA clones for both proteins by transient expression in mammalian cells using a high complexity human T cell cDNA library. COS-7 cells with be transfected with the library by DEAE-Dextran and cells expressing either p43 or p58 will be selected by """"""""panning"""""""" on antibody- coated plates. The plasmid DNA will be isolated from the adherent cells. After three rounds of transfection and selection, the cDNA insert(s) will be isolated and sequenced. Future goals of this work include the characterization of the physiological ligand(s) for the PTx receptor complex and the investigation of the role of this complex in non-T cells.
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