Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA.

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.

Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation.

TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons. TrkA expression in neuronal tumors also supports NGF-dependent differentiation of neuroblastomas and apoptosis of medulloblastomas. Phosphorylation of the activation loop tyrosines in RTK's are essential to activation as well as allosteric changes that facilitate substrate interaction and phosphorylation. Acidic amino acid substitution of the activation loop tyrosines in TrkA, Tyr683Tyr684, was performed to mimic the negative charges normally induced by ligand activation and receptor phosphorylation. A total of eight independent mutants containing single or double substitutions were generated for comparison. Herein, we demonstrate that acidic substitution of the activation loop tyrosines is sufficient to induce allosteric changes required for constitutive TrkA kinase activity as well as phosphorylation of TrkA signaling proteins such as Shc, PLCgamma-1, FRS-2 and erk1/2. The strongest constitutively active TrkA mutants, GluAsp and AspGlu, support NGF-independent neuritogenesis and cell survival to levels approximately 65 and 80-100%, respectively, of NGF-activated wild type TrkA. Thus, constitutively active TrkA may provide a useful strategy in future therapeutic approaches to limit the development and progression of neuronal tumors.

[Evaluation of dysarthria with the assistance of acoustic speech analysis in patients with amyotrophic lateral sclerosis]. / Ocena dyzartrii za pomoca analizy akustycznego sygnalu mowy u chorych z stwardnieniem bocznym zanikowym.

The aim of the study was to assess dysarthria in ALS subjects using acoustic speech analysis. The study was performed in 47 definite or probable ALS patients aged 29-76 years (mean age 53.7 yr.) and in 30 age and sex matched healthy control subjects. Neurological examination showed 15 dysarthric ALS subjects. Acoustic speech analysis is a quantitative, computer-acoustic method estimating dysarthria and based on assessing of sound distance from speech sound tests. In both group the mean sound distance between chosen sounds was compared to a basic pattern and was measured on time-frequency computer acoustic analyses (delta f = 125 Hz, delta T = 9 ms, delta s = 0.5 dB). Our results demonstrated that all sounds were incorrect in all ALS subjects. These abnormalities were significantly increased in the dysarthric ALS subjects. The mean sound distances which separated ALS from control subjects is 0.2 (by Euclidian principle) in 4 out of 5 measured sounds. We suggest that it is possible to detect and measure dysarthria in ALS patients based on the acoustic speech analysis, also in the limb onset ALS subjects.

[Etiopathogenesis of diabetic neuropathy]. / Etiopatogeneza neuropatii cukrzycowej.

The cause of diabetic neuropathy remains unclear but several mechanisms have been considered. Hyperglycemia with all its metabolic and vascular consequences probably plays the main role. Metabolic consequences of hyperglicemia including increased level of sorbitol and fructose, myoinositol deficiency and Na+/K+ adenosine triphosphate deficiency alter the function of peripheral nerves and lead to an oxidative stress, which changes fatty acid metabolism. Chronic hyperglycemia is also responsible for abnormalities in microcirculation that could lead to local ischemia evoking many endothelial changes. There are also some genetic factors, impaired neurotrophic support and autoimmune damage that influence sensitivity to peripheral nerve damage in diabetes mellitus.

The signaling adapter FRS-2 competes with Shc for binding to the nerve growth factor receptor TrkA. A model for discriminating proliferation and differentiation.

We have isolated a human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target and shown that it is tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, we demonstrate that the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclin-dependent kinase substrate p13(suc1), and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation.

A kinase insert isoform of rat TrkA supports nerve growth factor-dependent cell survival but not neurite outgrowth.

To investigate potential differences between the family of Trk receptors that might have differential consequences on cell signaling, we generated a rat TrkA homologue of the 14-amino acid kinase insert isoform of TrkC termed TrkAKi. Signal transduction by the TrkAKi receptor has been investigated and compared with the homologous signaling defective TrkC(Ki14) receptor. Herein, we demonstrate that TrkAKi receptors show a decrease in the absolute amount of kinase activity relative to wild-type TrkA, yet retain normal patterns of receptor tyrosine phosphorylation, as determined by phosphopeptide mapping studies, unlike TrkC(Ki14). nnr5 cell clones expressing TrkAKi receptors show a decrease in nerve growth factor (NGF)-mediated SHC tyrosine phosphorylation and a loss of high-affinity TrkA-SHC interaction comparable to those expressing TrkC(Ki14). Moreover, nnr5 cells expressing TrkAKi receptors fail to demonstrate NGF-dependent tyrosine phosphorylation of the signaling molecules phospholipase Cgamma-1, MAP kinase/ERK-1, and SNT. TrkAKi receptors internalize NGF comparable to wild-type TrkA, but do not stimulate neurite outgrowth. It is interesting that, unlike TrkC(Ki14), TrkAKi receptors retain phosphatidylinositol 3-kinase activity and nnr5 cells stably expressing TrkAKi receptors retain NGF-dependent cell survival under serum-free conditions. Lastly, TrkAKi receptors fail to stimulate three immediate-early genes (NGF1A, NGF1B, and c-fos), suggesting that these gene products are not required for NGF-dependent cell survival responses.