Structural and Functional Studies of the T Cell Antigen Receptor 
protein that associates with the f-chain; we are in 
the process of characterizing this protein. We 
would also like to understand the function of the 
associated CD3 complex within the complex 
oligomeric TCR. Moreover the ability to create 
such functional chimeric receptors may permit 
the creation of novel antiviral or antitumor TCRs 
that may be of value in gene therapy. 
Stimulation of the TCR initiates cellular activa- 
tion by inducing a transmembrane signal that is 
manifested as the formation of intracellular bio- 
chemical mediators called second messengers, 
which can initiate or influence cellular response 
pathways. Recent studies demonstrate that the 
TCR activates an uncharacterized protein-tyro- 
sine kinase (PTK) as the initial event leading to 
cellular activation. This PTK appears to be asso- 
ciated with the TCR f-chain. The activation of 
this PTK results in the tyrosine phosphorylation 
of many cellular proteins, one of which is phos- 
pholipase C-7I (PLC-7I). The phosphorylation 
of PLC-7I activates this enzyme to hydrolyze a 
rare membrane lipid, PIP2 (phosphatidylinositol 
4,5-bisphosphate). This yields two potent intra- 
cellular second messengers (inositol 1,4,5-tris- 
phosphate and diacylglycerol) that regulate the 
mobilization of intracellular calcium and activa- 
tion of the enzyme protein kinase C, respectively. 
These latter two events are physiologically im- 
portant to subsequent cellular responses. 
The mechanism by which the TCR couples to 
intracellular signaling pathways is largely unde- 
fined, as are many of the components of the sig- 
naling pathways themselves. To define and char- 
acterize the molecular basis by which the TCR 
regulates these pathways, we are using a somatic 
cell genetic approach. In work supported by a 
grant from the National Institutes of Health, we 
have isolated a number of mutants derived from T 
cell leukemic lines that are defective in TCR- 
mediated activation of the inositol phospholipid 
pathway. Unlike the parental cells, none of these 
mutants produce lymphokines in response to 
TCR stimulation. These mutants define four dis- 
tinct gene products other than the TCR chains 
that are required for the functional activation of 
the inositol phospholipid pathway. 
In one mutant, the activation of the tyrosine 
kinase pathway is still intact; however, some of 
the substrates of the tyrosine kinase pathway are 
not phosphorylated, including PLC-7I. The phe- 
notype of this mutant suggests that there may be a 
coupling protein or second PTK that is deficient. 
The defect in another of these mutants can be 
attributed to the absence of a cell surface protein, 
CD45, with tyrosine phosphatase activity. The 
absence of CD45 prevents the TCR from activat- 
ing the tyrosine kinase or phosphatidylinositol 
inositol pathway. At least one target of the CD45 
tyrosine phosphatase is a regulatory phospho- 
tyrosine residue in the PTK Ick. Thus a protein- 
tyrosine phosphatase regulates the activity of a 
PTK. This suggests a complex autoregulatory sys- 
tem that we are intensively studying. In the re- 
maining two mutants, biochemical studies have 
complemented our genetic approach. In neither 
of these mutants is the PTK pathway activated. 
Preliminary studies suggest that one of these mu- 
tants is deficient in a previously identified PTK. 
Thus these mutants are proving to be valuable 
tools with which to dissect the complexities of 
the signal transduction pathways and their rela- 
tionships to cellular responses. 
T cell activation is a complex process that is 
regulated by cell surface molecules. Investiga- 
tion of the molecules and events involved in the 
activation of T cells should lead to a more com- 
plete understanding of T cell biology and a more 
rational approach to the manipulation of the im- 
mune system. Moreover, through the study of the 
activation of T cells, it is likely that insight into 
other biological systems involving cell prolifera- 
tion and differentiation will emerge. 
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