Cyclic-AMP response element-based signaling assays for characterization of Trk family tyrosine kinases modulators.
Neurotrophins (NTs) induce gene transcription by binding their high-affinity tropomyosin-related kinase (Trk) receptors and initiating intracellular signal transduction cascades. In particular, activation of the cyclic AMP response element (CRE) in the promoters of target genes serves as surrogate markers for Trk receptor activation as demonstrated in both in vivo and in vitro systems. We used a HEK293 cell line stably expressing a CRE-luciferase reporter gene to develop an assay for monitoring Trk activation in response to their cognate ligands. Using TrkB, we showed that the assay was sensitive to physiological concentrations of brain-derived neurotrophic factor (BDNF) and that the signal was sufficiently robust to be suitable for implementation in high-throughput format. Further characterization of the TrkB expressing stable cell lines showed high-affinity binding for BDNF, a high density of receptor expression, and supported BDNF-mediated phosphorylation signaling. Consistent with this, inhibitors of phosphatidylinositol 3-kinase and the phospholipase C-gamma pathways led to reduction of BDNF-mediated luciferase responses. In contrast, inhibitors of mitogen-activated protein kinase pathways further potentiated BDNF responses. This assay was NT-Trk receptor pair-selective and shown to be further applicable to other Trk family members. This assay may be useful in screening compound libraries to identify Trk agonists, which may be applied towards discriminating between the activities of the different Trk receptor family members and the development of pharmacological drugs.
Zhang
Jie
J
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543-8000, USA.
Chen
Diana
D
Gong
Xiaohai
X
Ling
Huaiping
H
Zhang
Guoming
G
Wood
Andrew
A
Heinrich
Julia
J
Cho
Seongeun
S
D015153:Blotting, Western / Q000379:methods; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D026121:Indole Alkaloids; D008156:Luciferases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay / Q000379:methods; D020813:Receptor, trkB / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014162:Transfection / Q000379:methods
D019208:Brain-Derived Neurotrophic Factor; D002227:Carbazoles; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; C049985:staurosporine aglycone; D008156:Luciferases; D020813:Receptor, trkB