INTERACTIONS BETWEEN BRAIN-DERIVED NEUROTROPHIC FACTOR AND THE TRKB RECEPTOR - IDENTIFICATION OF 2 LIGAND-BINDING DOMAINS IN SOLUBLE TRKB BY AFFINITY SEPARATION AND CHEMICAL CROSS-LINKING

The extracellular domain of the human neurotrophin TRKB receptor expre ssed 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 prot eolytic 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 cysteine cl uster domain, The second domain is close to the second immunoglobulin- like domain (amino acid residues 342-394). Each of these two domains c an bind BDNF independently, Disulfide linkages present in the first do main are necessary for BDNF binding, probably because of preservation of the native conformation, To study the second domain in greater deta il, a truncated form of TRKB containing the second immunoglobulinlike domain (residues 248-398) was expressed in Escherichia coli, This doma in was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide coupling reaction. Several synthetic peptides correspon ding to amino acid residues 343-379 were able to bind immobilized BDNF , Amino acid substitution and cross-linking analysis indicated that am ino acids Phe(347), Asp(354), and Tyr(361) are intimately involved in BDNF binding, These results, obtained from a variety of experimental t echniques, highlight the importance of two distinct regions of the ext racellular domain of the TRKB receptor in binding BDNF.