The TrkB receptor protein-tyrosine kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. In response to brain-derived neurotrophic factor and neurotrophin-3 treatment, TrkB expressed exogenously in Rat-2 cells is rapidly phosphorylated on tyrosine residues. At least 2 regions of TrkB contain phosphorylated tyrosines. The major sites of autophosphorylation are in the region containing Tyr-670, Tyr-674, and Tyr-675, which lies in the kinase domain and corresponds by sequence homology to the Tyr-416 autophosphorylation site in p60c-Src. Tyr-785, which lies just to the COOH-terminal side of the kinase domain in a relatively short tail characteristic of the Trk family of protein-tyrosine kinase receptors, is also phosphorylated in response to neurotrophin-3 treatment. The sequence around Tyr-785 fits a consensus sequence for binding phospholipase C-gamma 1. The simplest interpretation of these results is that, in response to neurotrophin binding, at least two and perhaps all three of the tyrosines in the Tyr-670/674/675 region are autophosphorylated independently, and Tyr-785 is autophosphorylated in vivo. Following activation of TrkB, phospholipase C-gamma 1 is phosphorylated on Tyr-783, Tyr-771, and Tyr-1254. Phospholipase C-gamma 1 also forms a complex with TrkB in response to neurotrophin-3 treatment, consistent with the possibility that one of the TrkB autophosphorylation sites provides a binding site for the phospholipase C-gamma 1 SH2 domains, as is the case for other receptor protein-tyrosine kinases. We conclude that phospholipase C-gamma 1 is directly phosphorylated by TrkB. Since phosphorylation of Tyr-783 and Tyr-1254 results in activation of phospholipase C-gamma 1, we predict that neurotrophin-3 leads to activation of phospholipase C-gamma 1 following binding to TrkB in Rat-2 cells.

Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR gamma-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cgamma (PLCgamma). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5'-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR gamma-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation.

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitation experiments demonstrated insulin-dependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.

Streptolysin O-permeabilized pancreatic acini were used to study compartmentalization of Ca2+ signaling and Ca2+ pools. In these cells, the inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ channels could be activated by a number of agonists (carbachol, cholecystokinin, or bombesin) or by activation of the entire cellular phospholipase C pool with GTP gamma S. Surprisingly, each of the antagonists interacting with acinar cells inactivated the channels after stimulation with GTP gamma S. In addition, when permeabilized cells were stimulated with more than one agonist, any antagonist to the specific agonists employed inactivated the channels. The aberrant behavior of the antagonists in permeable cells was not related to a loss of specificity since (a) when added before GTP gamma S, the antagonists had no effect on Ca2+ release and (b) when cells were stimulated with a single agonist, the antagonists prevented only the effect of their specific agonist. The differential behavior of the antagonists in intact and permeable cells suggests a compartmentalization of Ca2+ signaling into separate, agonist-specific units that is modified by cell permeabilization. Further evidence for compartmentalization of signaling was obtained by showing that the partial agonist (the CCK octapeptide analogue JMV-180) can access and release only 50% of the cholecystokinin- or IP3-mobilizable Ca2+ pool in intact and permeable cells. Kinetic measurements revealed a multiphasic time course of agonist-evoked Ca2+ release in permeable cells. At high agonist concentrations, all phases were fast and merged into an apparent single event of Ca2+ release. The phases were separated by three independent protocols: reduction in agonist concentrations, addition of heparin, or addition of guanosine-5'-O-(thio)diphosphate. Since all protocols that caused phase separation reduce IP3-mediated Ca2+ release, these findings demonstrate heterogeneity in the affinity for IP3 of channels present in compartmentalized Ca2+ pools of the same cells. Compartmentalization of signaling and the heterogeneity in the affinity for IP3 resulted in a quantal agonist-evoked Ca2+ release. The overall findings are discussed in the context of an integrated model of compartmentalization of signaling complexes, Ca2+ pools, and IP3-activated Ca2+ channels.