OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

BACKGROUNDThe molecular mechanism of human tetralogy of Fallot (TOF) is incompletely defined. Animal models have suggested that neurotrophic tyrosine receptor kinase 3 (NTRK3) might be associated with the outflow tract defect, similar to that seen in human TOF, however, the expression pattern of NTRK3 in human TOF heart tissues has never been investigated.

METHODSQuantitative real-time PCR (qRT-PCR) and immunohistochemistry were applied to detect NTRK3 mRNA and protein levels in right ventricular outflow tract tissue samples of TOF patients, ventricular septal defect (VSD) patients and normal control infants (n = 10 in each group).

RESULTSqRT-PCR analysis indicated that NTRK3 mRNA levels were significantly decreased in the TOF group compared to the VSD group (0.024 +/- 0.003 vs 0.085 +/- 0.004, P = 0.022) and the normal control group (0.024 +/- 0.003 vs 0.091 +/- 0.002, P = 0.006). Quantitative immunohistochemical analysis showed that NTRK3 protein was mainly localized in the myocardium cytoplasm in all 3 groups. The immunoreactivity of NTRK3 protein was again significantly lower in the TOF group compared to the VSD group (1.42 +/- 0.62 vs 14.12 +/- 1.83, P = 0.023) and the control group (1.42 +/- 0.62 vs 16.25 +/- 2.31, P = 0.008). The expression of NTRK3 in the VSD group and in the control group showed no significant differences at both mRNA and protein levels.

CONCLUSIONSInsufficient expression of NTRK3 is associated with the outflow tract defect of human tetralogy of Fallot and may contribute to the progression of this defect.

Female ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Infant ; Male ; Receptor, trkC ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Temperature ; Tetralogy of Fallot ; genetics ; metabolism

Objective: To investigate the clinicopathological features and differential diagnosis of NTRK3 gene rearrangement thyroid papillary carcinoma (PTC). Methods: The PTC cases without BRAF V600E mutation were collected at Fujian Provincial Hospital South Branch from January 2015 to January 2020. The cases of NTRK3 gene rearrangement PTC were examined using immunohistochemistry and fluorescence in situ hybridization (FISH). The clinical data, histopathological characteristics, immunohistochemical features and molecular pathological changes were retrospectively analyzed. Data from the TCGA PTC dataset and the literature were also studied. Results: A total of 3 PTC cases harboring NTRK3 gene rearrangement were confirmed. All the patients were female, aged from 26,49,34 years. Histologically, two of them demonstrated a multinodular growth pattern. Only one case showed prominent follicular growth pattern; the other two tumors showed a mixture of follicular, papillary and solid growth patterns. All tumors showed a typical PTC nuclear manifestation, with some nuclear pleomorphism, vacuolated foci and oncocytic features. The characteristic formation of glomeruloid follicular foci was present in two cases which also showed psammoma bodies, and tumoral capsular or angiolymphatic invasion. The background thyroid parenchyma showed chronic lymphocytic thyroiditis. Mitotic rates were low, and no cases had any tumor necrosis. The pan-TRK and TTF1 testing was both positive in 3 cases, while S-100 and mammaglobin were both negative in them. FISH studies confirmed the NTRK3 gene rearrangement in all 3 cases. Studies on the TCGA datasets and literature revealed similar findings. Conclusions: NTRK3 gene rearrangement PTC is rare. It may be easily misdiagnosed due to the lack of histological and clinicopathological characteristics. Molecular studies such as pan-TRK immunostaining, FISH and even next-generation sequencing are needed to confirm the diagnosis. Immunohistochemistry of pan-TRK performed in the PTC cases without BRAF V600E mutation can be used as a good rapid-screening tool. With the emergence of pan-cancer tyrosine receptor kinase inhibitors, proper diagnosis of these tumors can help determine appropriate treatments and improve their outcomes.
Biomarkers, Tumor ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Mutation ; Proto-Oncogene Proteins B-raf/genetics* ; Receptor, trkC ; Retrospective Studies ; Thyroid Cancer, Papillary/genetics* ; Thyroid Neoplasms/genetics*

OBJECTIVETo study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.

METHODSYeast two-hybrid assay was used. The intracellular domains of TrkC and TrkC mutants were cloned into pAS2-1, and ShcD and its four domains (CH2, PTB, CH1, and SH2 domains) were cloned into pACT2 vector respectively. The constructs were separately cotransformed into yeast. beta-galactosidase activity was measured to detect their interactions. TrkC was cloned into pmRFP (carrying red fluorescent protein), and ShcD was cloned into pEGFP (carrying green fluorescent protein). pmRFP-TrkC and pEGFP-ShcD were co-transfected into 293T cells, and then the cells were fixed and subjected to confocal analysis to study their subcellular localization.

RESULTSShcD interacted with TrkC but not with kinase dead mutant TrkCM1(K572A). Both PTB and SH2 domains were capable of binding to TrkC, and PTB domain bound NPQY motif of TrkC. ShcD colocalized with TrkC throughout the cytoplasm and in the plasma membrane in 293T cells.

CONCLUSIONShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Binding Sites ; Cells, Cultured ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Protein Binding ; Receptor, trkC ; genetics ; metabolism ; Shc Signaling Adaptor Proteins ; genetics ; metabolism ; Transfection ; Transformation, Bacterial ; Two-Hybrid System Techniques ; src Homology Domains ; genetics

BACKGROUND AND OBJECTIVES: Neurotrophins have been known to be responsible for the differentiation and survival of developing neurons as well as for aiding the recovery of adult neurons from injury. The neurotrophin family includes NGF, BDNF, NT-3, and NT-4/5, and they exert their biological functions through activation of the high-affinity binding receptors, that is trkA, trkB, and trkC, with high characteristic specificity. Previous studies indicate that spiral ganglion cells express trkB and trkC mRNAs, while auditory hair cells produce NT-3 mRNA that directly affect maturation and survival of auditory neurons. It has been reported that the loss of target innervation and the eventual degeneration of auditory neurons caused by aminoglycoside ototoxicity can be prevented by the infusion of neurotrophic factors. The purpose of this study is to provide the expression patterns of trkB and trkC in the normal chochleas and damaged cochleas with aminoglycoside ototoxicity. MATERIALS AND METHODS: Adult Sprague-Dawley rats were treated with amikacin 500 mg/kg for ten days, and sacrificed on the 7th, 14th, 21th, and 28th day following the last injection. Auditory brainstem response was measured in each animal. Immunohistochemical method was used to study the localization of trkB and trkC receptors in the cochleas of adult rats of either normal control group or ototoxicity group. RESULTS: Immunoreactivities to trkB and trkC receptors were strongly positive in the spiral ganglion cells of all cochleas, especially in the neuronal perikarya of the type I cells. No difference in staining pattern was seen among the cochleas with different hearing thresholds. CONCLUSION: The uniform expression pattern of trkB and trkC receptors in the spiral ganglion cells regardless of the degree of ototoxicity suggests that neurotrophic factors may bind to these receptors to initiate the cellular mechanisms for neuronal survival in the injured auditory system.
Adult ; Amikacin ; Animals ; Brain-Derived Neurotrophic Factor ; Cochlea* ; Evoked Potentials, Auditory, Brain Stem ; Hair Cells, Auditory ; Hearing ; Humans ; Immunohistochemistry ; Nerve Growth Factor ; Nerve Growth Factors ; Neurons ; Rats* ; Rats, Sprague-Dawley ; Receptor, trkC* ; RNA, Messenger ; Sensitivity and Specificity ; Spiral Ganglion