MODULATION OF T CELL RECEPTOR (TCR) SIGNAL TRANSDUCTION BY CAMP

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To study the mechanism of activation of the hydrolysis of inositol phospholipids (InsPL) in response to perturbation of the murine T lymphocyte Aq receptor (TCR), and its regulation by activators of the adenylate cyclase (AC) system. The hydrolysis of InsPL, one of the most rapid responses observed in cells, is deemed to have a role as a transducer of the signal initiated at the TCR. This metabolic pathway is centered on the activation of an enzyme, a InsPL-specific phospholipase C (PLC). Little is known about the regulation and coupling mechanism of this enzyme with the TCR. A second signal transduction mechanism of relevance in T lymphocyte activation is represented by the AC/cAMP pathway. AC activation in Th cells has been clearly documented in response to either-pharmacological treatment (i.e. FSK, CTx) of lymphocytes or to autocoids (i.e.: prostaglandin E2). Increased levels of cAMP are associated with down regulation of several T cell responses, including lymphokine secretion and proliferation. The mechanism by which increased levels of cAMP depress lymphocyte function is not defined, but our preliminary data indicates that it is associated with a decrease in InsPL hydrolysis. we propose to investigate the effect of AC activators and cAMP on the regulation of InsPL hydrolysis in response to TCR perturbation in intact or permeabilized cells. This approach will allow us to characterize the site of action of cAMP in inhibiting TCR-mediated InsPL hydrolysis. We have been able to induce InsPL hydrolysis in permeabilized T cells by the addition of non-hydrolyzable analogs of GTP (such as GTP-gamma-S), which are otherwise non-permeable to intact cells. GTP-gamma-S activates a G-protein that regulates PLC activity, bypassing the TCR (or other cell surface structure), supporting the hypothesis of a role for G-proteins in TCR signal transduction. If the activation of the AC/cAMP/PKA pathway in this model results in inhibition of INSPL hydrolysis, then a direct effect of the cyclic nucleotide on PLC or on G-protein is likely. Alternatively, the failure of cAMP to inhibit under these experimental conditions will suggest an effect at a level proximal to G-protein activation, possibly at the level of the receptor.