Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor.

Abstract

Neurotrophin‐3 (NT‐3) has low‐affinity (Kd = 8 × 10(−10) M), as well as high‐affinity receptors (Kd = 1.8 × 10(−11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF), NT‐3 also binds to the low‐affinity NGF receptor, a molecule that we suggest to designate low‐affinity neurotrophin receptor (LANR). NT‐3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT‐3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT‐3 to its high‐affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand‐fold higher than those corresponding to their Kd to their high‐affinity neuronal receptors. Thus, specific high‐affinity NT‐3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed.

The Neurotrophin Receptor p75 Binds Neurotrophin-3 on Sympathetic Neurons with High Affinity and Specificity

“…Cell suspensions of neurons freshly isolated from E11 chick sympathetic ganglia were incubated with radiolabeled NT3 at 3 ϫ 10 Ϫ11 M, a concentration preferentially filling high-affinity NT3 binding sites (Rodríguez-Tébar et al, 1992). After cross-linking with the bifunctional hydrophilic cross-linker BS 3 , the neurotrophin receptor complexes were analyzed by gel electrophoresis and autoradiography.…”

“…NGF, N T3, and BDN F were iodinated using lactoperoxidase and 125 I (Amersham IMS30) as described previously (Rodríguez-Tébar et al, 1992;Dechant et al, 1993a). The specific radioactivity was 125-175 cpm /pg neurotrophin, and radiolabeled material was used within 1 week after the reaction.…”

“…This value is similar to the affinities of the NT3 high-affinity receptors on other neuronal populations (Rodríguez-Tébar et al, 1992) and for a proportion of the sites formed by recombinant trkC-expressing cell lines (Tsoulfas et al, 1993). trkC was not detectable in cultured neurons, however, indicating that this receptor cannot play a significant role in p75 N TR high-affinity binding, which therefore seems to be independent of the formation of a ternary complex with trkC.…”

Specific neurotrophin binding to leucine‐rich motif peptides of TrkA and TrkB

“…in terms of the underlying highly specific biochemical mechanism, the observed binding phenomena apparently can not quantitatively account for the binding affinity observed in vivo [3,22,25,34] or using recombinant receptor proteins [39]. It must therefore be assumed that the capacity of the columns was aggravated by the fact that a large percentage of the peptides was either coupled to the matrix in a way that abolished binding affinity or was simply folded in an incorrect fashion [4,10,20].…”

“…TrkA and TrkB share 37% amino acid sequence identity in the extracellular domains and 75% in the kinase domains [23,28]. Despite this high degree of similarity, TrkB is a much more versatile receptor than TrkA in that it binds three different ligands [24] with clearly distinguishable pharmacological characteristics [2,22,25,26] leading to distinct biological effects [8,14,15]. In order to show whether the second LRM also plays a role in ligand binding in the TrkB receptor, a peptide corresponding to this region (L2B) was synthesized and characterized in the same way as L2A.…”