Phospholipase C-␥1 (PLC-␥1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-␥1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-␥1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-␥1 GEM recruitment, but were required for PLC-␥1 phosphorylation at Tyr A trio of hematopoiesis-specific adaptors, including LAT, 3 Gads, and SLP-76, serve as pathway-specific regulators of phospholipase C-␥1 (PLC-␥1) (1). PLC-␥1 is a ubiquitous enzyme, regulated by phosphorylation at Tyr 775 and Tyr 783 (2-4) and involved in many different signaling pathways (5, 6). When activated, PLC-␥1 hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, which trigger calcium flux and contribute to protein kinase C and Ras activation, respectively. In T cells, the coordinated activity of calcium-and Rasdependent signaling pathways leads to the activation of NFAT, a regulator of interleukin-2 transcription (7,8). Given the importance of PLC-␥1-dependent signaling events, it is not surprising that the activation of PLC-␥1 is tightly regulated.The LAT, Gads, and SLP-76 adaptor proteins appear to constitute an integrated signaling unit that couples immunoreceptormediated activation of cytoplasmic tyrosine kinases to the activation of PLC-␥1 (1, 9). The use of three adaptor proteins to regulate one enzyme is a recurrent theme in complex signaling pathways. In a similar fashion, three adaptors (FRS2, Grb2, and Gab1) cooperate to activate phosphatidylinositol-3-kinase in the fibroblast growth factor receptor signaling pathway (10, 11). Evidence that LAT, Gads, and SLP-76 work in concert includes their similar expression patterns and localization to the same signaling complex; similar signaling defects observed in LATand SLP-76-deficient T cell lines; and similar T cell and mast cell phenotypes observed in LAT-, SLP-76-, and Gads-deficient mice (1,9,12). Most convincingly, SLP-76 alone cannot reconstitute B cell receptor signaling in a B cell line lacking the SLP-76 analog BLNK (B cell linker); rather, cotransfection of SLP-76, LAT, and Gads is required (13,14). Over the past years, the mechanisms whereby these adaptors mediate the activation of PLC-␥1 are beginning to be understood, as summarized briefly below.LAT, a transmembrane adaptor protein, is constitutively localized to specialized membrane microdomains known as glycosphingolipid-enriched membrane domains (GEMs), lipid rafts, or detergent-insoluble glycolipid-enriched membrane domains (15, 16). Upon T cell receptor (TCR) stimulation, LAT is heavily tyrosine-phosphorylated (17, 18), and a motif encompassing LAT phosph...

SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-␥1 (PLC-␥1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-␥1 (SH3 PLC ) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-␥1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCRinduced activation of PLC-␥1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-␥1 activation, we mapped the PLC-␥1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3 PLC , but is fully functional, mediating TCR-induced phosphorylation of PLC-␥1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3 PLC to activate PLC-␥1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.