A novel mutation within the extracellular domain of TrkA causes constitutive receptor activation

Abstract

The TrkA NGF receptor extracellular region contains three leucine repeats¯anked by cysteine clusters and two immunoglobulin-like domains that are required for speci®c ligand binding. Deletion of the immunoglobulinlike domains abolishes NGF binding and causes ligand independent activation of the receptor. Here we report a speci®c mutation that increases the binding a nity of the TrkA receptor for NGF. A change of proline 203 to alanine (P203A) in the linker region between the leucine repeats and the ®rst Ig-like domain increased NGF binding by decreasing the ligand rate of dissociation. This mutated receptor was appropriately expressed on the cell surface and promoted ligand-independent neurite outgrowth in PC12nnr5 cells. The mutant receptor was capable of spontaneous dimerization and was constitutively phosphorylated in the absence of ligand. Moreover, expression of TrkA-P203A receptor in ®broblasts induced DNA synthesis and transformation and generated tumours in nude mice. These data suggest that domains outside of the immunoglobulin-like structure contribute to ligand binding and constitutive activation of Trk receptors. Oncogene (2001) 20, 1229 ± 1234.

An Intracellular Domain Fragment of the p75 Neurotrophin Receptor (p75NTR) Enhances Tropomyosin Receptor Kinase A (TrkA) Receptor Function

“…Structural and biochemical data indicate that ligand binding to TrkA at these sites can be allosterically modulated and facilitated by p75 NTR through the additional surface exposure of the more N-terminally located IgG-C1/LRM domains (51). Although this may occur due to mutation of residues within these N-terminal domains, such as occurs in the TrkA P203A mutant used herein (32), the extracellular domain of wild-type TrkA is considered to be rigid. Thus, the structural change required to enable this modulation in vivo is probably mediated via the TrkA intracellular and/or transmembrane domains (16).…”

“…Finally, we examined the ability of c29 to modulate NGF binding to a TrkA variant (TrkA P203A ), which has a constitutively increased binding affinity for NGF due to a mutation within the extracellular flexible linker region (32). c29 had a negligible effect on the rate of NGF binding to HEK293 cells expressing TrkA P203A (Fig.…”

“…8b). One mechanism by which receptor ligand-binding capacity can be increased without changing receptor numbers is through structural modulation of the ligand-binding site (32,51). To investigate whether c29 might be causing a conformational change to facilitate the access of ligands to their binding site within the extracellular domain of TrkA, we compared the extent of binding of a TrkA antibody that targets the NGF ligand-binding site (AF1056), versus a non-modulatory TrkA antibody (06-574), to PC12 cells treated with c29.…”

p75 Co-receptors Regulate Ligand-dependent and Ligand-independent Trk Receptor Activation, in Part by Altering Trk Docking Subdomains

“…TrkA IgG-C1 deletions and a mutation also show constitutive activation (36,37), as does a Cys to Ser substitution in the IgG-C2 subdomain (38). In addition, others proposed a role for the IgG-C1 subdomain in neurotrophin binding (7,9).…”

TrkA Receptor “Hot Spots” for Binding of NT-3 as a Heterologous Ligand

“…Moreover, it has also been shown that the D4 subdomain has a role in regulating receptor dimerization (17) and in constitutive activation of the receptors (18). The D1 subdomain of TrkA can be utilized by NGF to activate the receptor, through a potentially allosteric or conformational mechanism regulated by co-expression of p75 on the cell surface (18).…”