The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein (␤APP). ␤APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of ␤APP leads to the secretion of A␤, the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of ␤APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. Protein-protein interactions are important in signaling by many cell surface receptors. For example, activated growth factor receptors bind several signaling proteins in a phosphotyrosine-dependent fashion. Many of these signaling proteins contain Src homology 2 (SH2) domains that mediate the interaction with growth factor receptors by binding to specific phosphopeptide sequences present on the receptors (40, 45). Our laboratory has been cloning receptor-binding proteins by screening bacterial expression libraries with the tyrosine-phosphorylated epidermal growth factor (EGF) receptor. In general, this approach has led to the cloning of genes that encode SH2-domain proteins (33,36,39,47). However, recently, our group and others have identified a new phosphotyrosine interaction/phosphotyrosine binding (PI/PTB) domain in the protein Shc (3, 23, 28). Shc has an important role in tyrosine kinase signal transduction. Shc becomes tyrosine phosphorylated upon activation of many growth factors, cytokines, and G-protein-coupled receptors (12,14,32,41,43,55). The tyrosine phosphorylation of Shc allows it to bind Grb2, which begins a cascade leading to the activation of Ras and mitogenactivated protein kinase pathways (43). The PI/PTB domain found in the amino terminus of Shc appears to be crucial for the interaction of Shc with EGF receptor, nerve growth factor receptor, and insulin receptor (1,3,13,16,52,54).Although both PI/PTB domains and SH2 domains bind to phosphotyrosine-containing peptides, their binding specificities and tertiary structures are completely different (48,56,59). While SH2 domain binding specificity is determined by residues that lie carboxy terminal to the phosphotyrosine, the Shc PI/PTB domain specificity is determined by residues amino terminal to the phosphotyrosine. The binding specificity for the Shc PI/PTB is the sequence ⌿XNPXpY (where ⌿ is a hydrophobic residue, N is asparagine, P is proline, X...

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. We analyzed the nine Drosophila Toll proteins and found that wild-type Toll-9 behaves similar to gain-of-function Toll-1. Toll-9 activates strongly the expression of drosomycin, and utilizes similar signaling components to Toll-1 in activating the antifungal gene. The predicted protein sequence of Toll-9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll-9. The Toll-9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll-9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms.

To identify receptor-associated proteins that may contribute to the specificity of insulin and IGF-I signaling responses, a mouse embryo library was screened using the yeast two-hybrid system. Multiple receptor-interactive clones encoding the SH2 domain of the adapter protein Grb10 were isolated. Subsequent cloning of the full-length Grb10 sequence from a mouse fat cDNA library defined a previously unknown Grb10

The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosinebinding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to receptor tyrosine kinases. We have generated homologous mutations in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. Our data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions.