Perturbation of the T cell antigen receptor (TCR) by antigen/MHC or by anti-receptor antibodies initiates a cascade of events which includes protein tyrosine kinase activation and the phosphorylation and activation of phospholipase Cgamma1 (PLCgamma1). The activation of PLCgamma1 is critical in that PLCgamma1-mediated phosphoinositide (PI) hydrolysis controls calcium mobilization and protein kinase C activation, two obligatory events in lymphokine secretions and cell proliferation. The objective of this project is to establish the mechanisms by which immune receptors regulate PLCgamma1. PLCgamma1 includes two src-homology (SH) 2 domains that bind tyrosine-phosphorylated proteins and an SH3 domain that interacts with proline-rich polipeptides. By acting as docking sites for other molecules, these domains play a role in coupling proteins to surface receptors or other regulatory molecules. Site-specific mutant constructs of an epitope-tagged ectopically-expressed PLCgamma1 protein were used to characterize structural requirements for PLCgamma1 activation, to identify proteins that interact with PLCgamma1 subdomains, and dissect the individual role of these domains in PLCgamma1 activation. These studies have shown that the amino terminal SH2 (SH2(N)) domain, but not the carboxyl-terminal SH2 (SH2(C)) domain, was required and sufficient for TCR-induced PLCgamma1 tyrosine phosphorylation. The defective phosphorylation of the SH2(N) domain mutant was associated with its inability to interact with a 36-38-kDa phosphoprotein. Further structural/ functional characterization of PLCgamma1 was accomplished by expressing wild type or mutant PLCgamma1 proteins in a PLCg-negative chicken B cell line, P10-14. Similar to our previous findings in T cells, the SH2(N) domain was required and sufficient for B cell receptor (BCR)-induced PLCgamma1 tyrosine phosphorylation. The association of PLCgamma1 with an 80-kDa tyrosine-phosphorylated protein was also exclusively dependent upon an intact SH2(N) domain. TCR or BCR-induced membrane translocation of PLCgamma1 required the SH2(N) domain, but was also decreased by mutation of the SH3 domain. The addition of a myristoylation signal to force membrane targeting of PLCgamma1 did not bypass the SH2(N) domain requirement for phosphorylation. BCR-induced PI hydrolysis was abrogated by mutation of either the SH2(N) or the SH2(C) domain and was partially decreased by mutation of the SH3 domain. BCR-induced activation of an NF-AT reporter gene in PLCgamma1-reconstituted P10-14 cells, an indication of Ca2+ mobilization, showed an absolute requirement for both the SH2(N) and SH2(C) domains, but not the SH3 domain. These data suggest a model of Ag receptor-induced PLCgamma1 activation whereby the SH2(N) domain is required and sufficient for promoting the interaction with a tyrosine kinase, while both the SH2(N) domain and the SH3 domain participate in translocating PLCgamma1 to the membrane. Tyrosine phosphorylation and membrane recruitment, however, are per se insufficient for the enzymatic activation of PLCgamma1, which also needs a functional SH2(C) domain. Future efforts will be directed at investigating the structural requirement for PLCgamma1 regulation via activation by other surface molecules (e.g., CD2, CD28) and their signaling cross-talk with the TCR.
