Abstract
We analyzed the function of Trk nerve growth factor (NGF) receptors containing a point mutation (Tyr-->Phe) in a major autophosphorylation site (Tyr-785). Tyr-785 is required for phospholipase C-gamma 1 to interact with Trk and to become tyrosine-phosphorylated in response to NGF. The altered receptors were transfected into a mutant subline of PC12 rat pheochromocytoma cells (designated PC12nnr5) that, unlike wild-type PC12 cells, lack expression of endogenous Trk and responsiveness to NGF. PC12nnr5 cells permanently transfected with Trk Y785F exhibit NGF-dependent autophosphorylation and normal NGF binding and internalization. Moreover, Trk Y785F mediates NGF-stimulated neurite outgrowth as well as a variety of additional responses including induction of immediate-early and late genes. However, in contrast to cells expressing wild-type Trk, cells expressing Trk Y785F lack NGF-promoted elevation of peripherin intermediate filament mRNA and protein. These observations indicate that phospholipase C-gamma 1 activation or other signaling pathways dependent on Tyr-785 autophosphorylation are selectively required for regulation of peripherin expression by NGF, but not for many other functional NGF responses. This supports the presence of multiple and separable signaling pathways in the NGF mechanism of action.
Highlights
We analyzed the functionof Trk nerve growth factor cellular proteins (generally those containing (SsrHc2homology (NGF) receptors containing apointmutation (Tyr + 2) domainsw) ith specific phosphotyrosine-containing se
The presence of multiple phosphotyrosine-containing mutantsubline ofPC12 ratpheochromocytoma cells sequences on receptors and of multiple SH2 domain signaling that, unlike wild-tyPpCe12 cells, molecules leads to activation of signaling cascades that are lack expression of endogenous Trk and responsiveness responsible for generation of diverse biological reto NGF.PC12nnr5 cells permanently transfected with sponses
We examined the ability of Trk Y785F to mediate effi
Summary
Vol 269,No.12,Issue of March 25,pp. 89011-9899410, Printed in U.S.A. Affecting Interaction with PhospholipasCe-yl Abolishes NGF-promoted Peripherin Induction but Not Neurite Outgrowth*. Transfection of NGFl binds t o the Trk receptor tyrosine kinase [1,2,3], an PCl2nnr cells with wild-type trk cDNA appears to restore all interaction required for functional responses to NGF [4]. We have as- an increasing gradient of acetonitrile in 0.5% aqueous trifluoroacetic sessed the functional activities of NGF in PCl2nnr cells that express a specific mutant form of Trk which no longer interacts with PLC-yl We find that these cells display apparently normal NGF binding properties and a wide variety of differentiation-associated NGF responses. Vectors for Cell lzansfection-Wild-type and mutant trk inserts were I n Vitro Autophosphorylation a n d Protein Kinase Assays-Proteins excised with EcoRI from the mutagenesis vector pKS and ligated into were immunoprecipitated with anti-Trkas described above, expression vector pCMV-IRV [13].Orientationwas confirmed with and the immunoprecipitates were washed three times with lysis buffer. Reverse Phase High Performance Liquid Chromatography a n d Ed-
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