Signal transduction events mediated by the BDNF receptor gp 145trkB in primary hippocampal pyramidal cell culture

Abstract

The trkB gene encodes a tyrosine kinase receptor, gp145trkB, for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). To understand the role of gp145trkB in the nervous system, we have investigated its expression in embryonic rat hippocampal pyramidal cell cultures and examined the effects of BDNF on signal transduction in the primary neurons. The expression of trkB transcripts was established by PCR analysis and in situ hybridization. In addition to gp145trkB, the pyramidal neuronal cultures expressed transcripts specific for the NT-3 receptor gp145trkC, but not for the high-affinity NGF receptor gp140trk or for p75LNGFR, a low-affinity receptor for all known members of the NGF family of neurotrophins including the gp145trkB ligands, BDNF and NT-4. The presence of gp145trkB receptors in the primary neuronal cultures was confirmed by immunocytochemical analysis in which > 90% of the cells stained with affinity-purified polyclonal antibodies to gp145trkB. Immunoblots using this antibody revealed a single approximately 140 kDa protein in both adult hippocampus and pyramidal cultures. Addition of recombinant BDNF to these cultures induced the tyrosine phosphorylation of gp145trkB, as determined by antiphosphotyrosine staining of gp145trkB immunoprecipitates. Moreover, BDNF treatment activated the microtubule-associated protein (MAP) kinases, as determined by an increase in MAP2 phosphorylation in vitro. Both the 41 and 44 kDa forms of MAP kinase were activated by BDNF. BDNF also increased c-fos expression in over 90% of the cells. These results indicate that gp145trkB does not require p75LNGFR to form a functional receptor for BDNF in hippocampal pyramidal neurons.