Interactions between Brain-derived Neurotrophic Factor and the TRKB Receptor

Abstract

The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein, possessing binding ability for brain-derived neurotrophic factor (BDNF). Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF. One of these domains consists of amino acid residues 103-181 and contains both the third leucine-rich motif and the second cys-teine cluster domain. The second domain is close to the second immunoglobulin-like domain (amino acid residues 342-394). Each of these two domains can bind BDNF independently. Disulfide linkages present in the first domain are necessary for BDNF binding, probably because of preservation of the native conformation. To study the second domain in greater detail, a truncated form of TRKB containing the second immunoglobulin-like domain (residues 248-398) was expressed in Esche-richia coli. This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiim-ide coupling reaction. Several synthetic peptides corresponding to amino acid residues 343-379 were able to bind immobilized BDNF. Amino acid substitution and cross-linking analysis indicated that amino acids Phe 347 , Asp 354 , and Tyr 361 are intimately involved in BDNF binding. These results, obtained from a variety of experimental techniques, highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF. The trk locus was first identified as a transforming gene in a human colon carcinoma biopsy (1-3). This locus encodes a cell surface receptor of the tyrosine kinase family (4). The TrkA gene product is a prototype for a subfamily of tyrosine kinase receptors including TrkB and TrkC (5-7). The Trk family of tyrosine kinases acts as functional receptors for the neurotro-phins: nerve growth factor, brain-derived neurotrophic factor (BDNF), 1 neurotrophin-3 (NT-3), NT-4/5, and probably NT-6 (7-11). Although the overall sequence homology between the extra-cellular domains of the Trk receptors is approximately 60%, each receptor specifically binds a subset of the neurotrophins. Recently, two distinct domains in the Trk receptors have been reported to contain neurotrophin binding sites. One region is the LRM domain of the TRK receptor as determined from expression of an unfolded fusion protein encoding the LRM region in Escherichia coli (12, 13). Analysis of a series of extra-cellular deletions and TRK chimeras indicated that the second Ig-like region was important for both neurotrophin binding and receptor activation (14, 15). In particular, these studies showed that the second Ig-like domain confers specificity and provides the main contacts for binding to Trk receptors by their ligands. Concurrently, receptor binding sites in the neurotrophins have been investigated by site-directed mutagenesis (for review , see Ref. 16). Although loop V (residues 94-98) of the neurotrophins is commonly thought to be the receptor binding region, extensive site-directed mutagenesis studies on NT-3 indicated that amino acid residue Arg 103 affected biological activity significantly (17, 18). In another study, small mono-meric cyclic peptide analogs that mimic the-turn regions of nerve growth factor were synthesized, and one region (amino acid residues 92-96) exhibited competitive inhibition and affected the biological response of treated cells (19). Recently, in the erythropoietin-erythropoietin receptor system, small Cys-containing peptides consisting of 20 amino acids were shown to dimerize the receptor (20). In total, these reports encouraged us to study the binding properties of small peptide fragments of TRKB to BDNF to identify critical amino acid residues. A variety of chemical approaches has been used to investigate receptor-ligand interactions or protein-protein interactions with immobilized ligand on membrane filters or agarose beads (21-25). MALDI-mass spectrometry in combination with proteolytic protection assays has been used to identify functional epitopes (24). The affinity-based approach has been used for chemical libraries prepared on beads or pins but has rarely been used in screening a library in solution or natural product extracts. One example of such an approach was the determination of which interleukin-6 peptide fragments interacted with its receptor using dot analysis on immobilized ligand-bound membrane (21). However, this approach was useful only when the primary structure alone contributed to most of the receptor-ligand interactions. Moreover, nonspecific binding of some particular peptides on agarose or Sepharose beads can occur, thus complicating analysis. Therefore, it is desirable to