Interactions between brain-derived neurotrophic factor and the TRKB receptor. Identification of two ligand binding domains in soluble TRKB by affinity separation and chemical cross-linking.
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 cysteine 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 Escherichia coli. This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 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 Phe347, Asp354, and Tyr361 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.
Haniu
M
M
Departments of Protein Structure, Protein Chemistry, and Immunology, Amgen, Inc., Thousand Oaks, California 91320, USA. mhaniu@amgen
Montestruque
S
S
Bures
E J
EJ
Talvenheimo
J
J
Toso
R
R
Lewis-Sandy
S
S
Welcher
A A
AA
Rohde
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism* / Q000494:pharmacology; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003432:Cross-Linking Reagents; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D019208:Brain-Derived Neurotrophic Factor; D003432:Cross-Linking Reagents; D008024:Ligands; D010446:Peptide Fragments; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases