Axel Obermeier

Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma.

Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal‐transducing proteins such as phospholipase C‐gamma (PLC gamma), phosphatidylinositol‐3′‐kinase (PI3′‐K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain‐containing substrates within the Trk cytoplasmic domain suggests a non‐essential function of PI3′‐K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co‐operative function of the PLC gamma‐mediated pathway for neuronal differentiation of PC12 cells.

Tyrosine 785 is a major determinant of Trk--substrate interaction.

Interaction of the nerve growth factor (NGF) receptor/Trk with cellular substrates was investigated by transient co‐overexpression in human 293 fibroblasts using ET‐R, a chimeric receptor consisting of the epidermal growth factor receptor (EGF‐R) extracellular ligand binding domain and the Trk transmembrane and intracellular signal‐generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C gamma (PLC gamma), ras GTPase‐activating protein (GAP) and the non‐catalytic subunit of phosphatidylinositol‐3′‐kinase, p85, in a ligand‐dependent manner. Deletion of 15 C‐terminal amino acids, including tyrosine 785 (Y‐785) abrogated receptor and substrate phosphorylation activities. Mutation of Y‐785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET‐YF phosphorylation of PLC gamma was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine‐phosphorylated synthetic C‐terminal peptide competitively inhibited Trk cytoplasmic domain association with PLC gamma. Thus, the short C‐terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y‐785 is a major and selective interaction site for PLC gamma.

Novel functional interactions between Trk kinase and p75 neurotrophin receptor in neuroblastoma cells.

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET‐R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET‐R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF‐induced response, leading to differentiation of almost all cells. A mutated NGF, 3T‐NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF‐induced differentiation required at least nanomolar concentrations of NGF, consistent with the low‐affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol‐3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET‐R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase‐3. An anti‐p75 antibody blocked the survival effect of NGF. Brain‐derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.