The low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation.

Abstract

Protein tyrosine phosphorylation is a potential mechanism for initial sinaling in PC12 cells during dfferentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of highaffinity NGF binding sites. In contrast to transfection of wildtype low-affinity p75 NGF receptors, transfection of p75NGm with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NG is involved in initiating pbosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosne kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation.Protein tyrosine phosphorylation is initiated by growth factor binding to receptors for insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), all of which possess intrinsic tyrosine kinase activity (1). Nerve growth factor (NGF) interacts with a 75-kDa receptor (2) that lacks a cytoplasmic tyrosine kinase domain (3,4), yet rapid and transient protein tyrosine phosphorylation has been observed upon treatment of PC12 cells with NGF (5-8). The kinase responsible for this activity has been identified as the trk protooncogene, a receptor tyrosine kinase that binds NGF and is activated in PC12 cells, neuroblastoma cells, and embryonic sensory neurons (8,9,38 Constructs. Construction of the deletion mutants of the NGF receptor has been described (15). All cDNAs were subcdoned into the EcoRI site of the pMV7 expression vector and recombinant retroviruses produced as described (18). Retroviral Gene Transfer. Cell lines expressing the wildtype and mutant NGF receptor cDNAs were generated after retroviral infection of helper-free virus stocks. NR18 transfectants were isolated after neomycin (0.5 mg of G418 per ml; GIBCO) selection and purification by rosetting with the monoclonal antibody ME20.4 and rabbit anti-mouse IgGcoupled human erythrocytes (2,3,15).Immunoblotting. Cells were grown to near confluence on 60-mm tissue culture dishes and were serum starved for 1 hr by incubation in serum-free DMEM. The medium was then replaced with DMEM containing 50 ng of NGF per ml or 20 ng of EGF per ml (Bioproducts for Science, Indianapolis). At the indicated times the cells were rapidly rinsed with ice-cold Tris-buffered saline, solubilized in 120 ,l of sol buffer [10 mM Tris hydrochloride, pH 7.4/1% sodium dodecyl sulfate (SDS)/1 mM sodium orthovanadate/0.1 mM sodium molybdate/1 mM phenylmethylsulfonyl fluoride/5 ,ug of aprotinin per ml/5 ,ug of leupeptin per ml], and immediately frozen in dry ice and ethanol. The samples were then boiled for 5 min. Protein concentrations were determined with a protein assay kit (Bio-Rad). Proteins (150 ,ug) were separated by SDS/ PAGE on 6-13% linear gradient polyacrylamide gels (unless otherwise noted) and electro...

Chimeric tumor necrosis factor-TrkA receptors reveal that ligand-dependent activation of the TrkA tyrosine kinase is sufficient for differentiation and survival of PC12 cells.

“…This view is supported by transfection studies, in which introduction of p75NGPR restores high-affinity binding in mutant cells lacking this protein and also allows NGF-induced protein phosphorylation (12)(13)(14). On the other hand, attempts at crosslinking the two receptors have so far failed (15), and they do not coimmunoprecipitate (9, 16).…”

Modeled structure of the 75-kDa neurotrophin receptor

“…Association with gpl40trk subunits has been highly controversial despite evidence of participation by NTR in high-affinity complexes (Berg et al, 1991;Hempstead et al, 1991;Battleman et al, 1993;Benedetti et al, 1993;Barker & Shooter, 1994;Hantzopoulos et al, 1994;Mahadeo et al, 1994;Wolf et al, 1995). Failure of chemical crosslinking to reveal a physical complex between NTR and gpl40trk (Meakin & Shooter, 1991) and a report that gpl40trk homodimers account for NGF signaling (Jing et al, 1992) have suggested that NTR does not form a heterodimer with gpl4Otrk.…”

Dual role for p75NTR signaling in survival and cell death: Can intracellular mediators provide an explanation?

“…In PC12 cells, NGF binding to p75 NTR enhances NGF-TrkA binding, TrkA autophosphorylation, and biological responses of TrkA (Berg et al, 1991;Barker and Shooter, 1994). p75 NTR signaling may also suppress certain aspects of TrkA activation required for neurite outgrowth.…”