Graves, J. D.; (1991) The role of protein in T lymphocyte activation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

An analysis of the role of guanine nucleotide-binding proteins in T lymphocyte activation. Proteins which bind and hydrolyse GTP are involved in the regulation of many aspects of cellular growth control and metabolism. For example, the heterotrimeric G proteins are involved in transducing signals from receptors to effect changes in cellular second messenger systems. Another class of guanine nucleotide binding proteins (p21ras) are encoded by the ras protooncogenes and have been implicated in the regulation of normal cell growth and oncogenic transformation. Using a permeabilised cell system which allowed cellular access for otherwise membrane-impermeant nucleotide and peptide reagents, while maintaining many intracellular signalling pathways, the role of guanine nucleotide binding proteins in T cell antigen receptor (TCR/CD3) complex coupling to phospholipase C (PLC) and in the regulation of protein kinase C (PKC) activity were investigated. In addition, the regulation of p21ras by T cell surface receptors was analysed in both intact and permeabilised cells. The data show that the TCR/CD3 complex and a population of G proteins can regulate PI-PLC in T cells but that the effects of guanine nucleotides on TCR/CD3 coupling are not compatible with a simple receptor G protein PI-PLC model. These results most likely reflect that the TCR/CD3 complex is not coupled to PLC via a G protein but that a guanine nucleotide binding protein can indirectly modulate TCR/CD3 coupling. An alternative TCR/GD3 coupling mechanism is suggested. Receptor and G protein agonists were also observed to induce PKC- mediated phosphorylation of the CD3 y subunit in permeabilised T lymphoblasts. These results are consistent with stimulation of PKC activity being explained by agonist effects on phosphatidylinositol metabolism. However, the ability dissociate G protein agonist effects on PI-PLC activity and phosphorylation of CD3 suggest that guanine nucleotide binding proteins also exert a regulatory effect on PKC activity by distinct mechanisms. This study also demonstrates that the activation state of endogenous p21 can be regulated by triggering the TCR/CD3 complex, CD2 antigen or the high affinity receptor for the growth factor IL2. These results infer that normal p21ras functions in the signalling pathways by which these receptors regulate T lymphocyte activation and proliferation. Evidence is provided to suggest that the TCR/CD3 complex regulates the GTPase activity of p21ras and that both PKC-dependent and independent pathways exist for the regulation of p21ras in T lymphocytes.

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