TrkA cross-linking mimics neuronal responses to nerve growth factor.

Abstract

TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that 297% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent antitrkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.

Expression of Neurotrophins and their Receptors in Human Bone Marrow

“…For TrkA (763), TrkB (794), and TrkC (798), specific controls were performed on adjacent slides, either by omitting the primary or secondary antibody or by coincubation with either the corresponding homologous control peptide or the heterologous control peptide (Santa Cruz Biotechnology) as previously reported. 38,39 Trk (C-14), TrkA (763), TrkB (794), and TrkC (798) IR were not altered by coincubation with the heterologous peptides and the Trk homologous peptide specifically abolished the signal.…”

Structure‐Activity Relationships of Conformationally Constrained Peptide Analogues of Loop 2 of Brain‐Derived Neurotrophic Factor

“…We predict that small dimeric loop 2 analogues may act as agonists if they can facilitate dimerization of trkB based on observations made in the highly analogous NGF-trkA receptor system. It has been shown that divalent antibodies to trkA can cause the homodimerization of this receptor, leading to signal transduction and NGF-like biological activity in vitro (Clary et al, 1994). It is reassuring that a small peptide mimetic of erythropoietin, produced by a recombinant library technique, possesses full erythropoietin-like biological activity by self-associating to form a dimer that dimerizes the erythropoietin receptor .…”

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

“…Then 10 g 1088 (IgG), 10 g anti-rat TrkA (RTA; ref. 72; IgG) or 5 g anti PLC-␥ mixed monoclonal (Upstate Biotechnology, Lake Placid, NY) was added, incubated overnight and recovered with protein-A-or protein-A͞G Sepharose (Pierce). Sepharose beads were washed twice in IP buffer and once with water, then resuspended in 50 l 7 M urea sample buffer (7 M urea͞2% SDS͞125 mM Tris, pH 6.95͞20 mM DTT͞0.1% Bromophenol blue) and heated (55°C for 15-30 min).…”