The objective of this study is to determine the coupling mechanism between the T cell receptor (TCR) of T helper (Th) lymphocytes and phospholipase C (PLC), the key enzyme in the inositol phospholipid (InsPL) hydrolysis pathway. An understanding of the mechanism of lymphocyte activation represents the base for designing immunomodulatory strategies targeted to critical elements of the signal transduction pathway, based upon the information acquired. Preliminary findings: Effect of quanine nucleotide analogs. Exposure of murine Th cells permeabilized with streptolysin O (SLO) or tetanolysin (TL) to the non-hydrolyzable guanosine triphosphate (GTP) analog, guanosine-5'-O- (3-thiotriphosphate) (GTP-gamma-S) , resulted in InsP generation. Similarly, perturbation of the TCR with the MoAb 145.2Cll (directed against the TCR CD3 epsilon chain) resulted in InsPL hydrolysis by permeabilized cells. A role for a G-protein in TCR/PLC coupling was further indicated by the inhibition of TCR-mediated InsPL hydrolysis by GDPBS, a guanine nucleotide analog that competes for GTP. Microelements and nucleotide requirement. InsP generation induced by either TCR perturbation or GTP-gamma-S treatment showed similar Ca(2+) dependence. ATP was strictly required for TCR-mediated INSPL hydrolysis, and potentiated GTP-gamma-S-induced InsP generation. Other nucleotides (CTP, GDP, GTP, ITP) did not affect the response. Effect of bacterial toxins that covalently modify G-proteins. Pertussis toxin (PTx) is a bacterial toxins that covalently modifies many G-proteins by ADP-ribosylating their alpha subunit. This modification prevents dissociation of the G-protein heterotrimer and blocks it from exerting its normal receptor coupling function. PTx treatment did not affect the stimulation of InsP production by perturbation of the TCR or GTP-gamma-S in intact or permeabilized cells, respectively. In the future we will attempt initial identification of this protein(s) by testing its cross-reactivity with antisera against amino acid sequences of known G-proteins. Particular attention will be dedicated to conserved sequences (i.e. GTP-binding site) and amino-terminal or carboxy-terminal sequences.
