MODULATION OF TCR /CD3 SIGNAL TRANSDUCTION BY C-AMP /PROTEIN KINASE A

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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 Ag receptor (TCR/CD3), and its regulation by activators of the adenylate cyclase (AC) system. The hydrolysis of InsPL, one of the most rapid responses observed in T cells, is deemed to have a role as a transducer of the signal initiated at the TCR/CD3. This metabolic pathway is centered on the activation of an enzyme, an InsPL-specific phospholipase C (PLC). 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/CD3 perturbation in intact or permeabilized cells. This approach will allow us to characterize the site of action of cAMP in inhibiting TCR/CD3-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 GTPgammaS), which are otherwise non-permeable to intact cells. GTPgammaS activates a G-protein that regulates PLC activity, thus bypassing the TCR/CD3 (or other cell surface structure). Activation of the cAMP/PKA pathway results in inhibition of InsPL hydrolysis triggered via TCR/CD3 perturbation, but does not affect GTPgammaS-induced PLC activation. Therefore the two pathways may be distinct. Although cAMP-mediated inhibition does not occur after permeabilization, addition of purified PKA reconstituted the inhibitory effect, which was associated with a specific pattern of phosphorylation of membrane proteins, and included a PLC isozyme, PLC-gamma1. Attempts will be made to understand the functional relationship between PLC- gamma1 phosphorylation by PKA and CD3-mediated activation.