NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobulin-like domains.
Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric receptors was constructed using cDNA sequences from rat trkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of trkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the trkA receptor.
Pérez
P
P
Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas, Universidad de Salamnca, Spain.
Coll
P M
PM
Hempstead
B L
BL
Martín-Zanca
D
D
Chao
M V
MV
D001483:Base Sequence; D002478:Cells, Cultured / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003627:Data Interpretation, Statistical; D015870:Gene Expression / Q000502:physiology; D007136:Immunoglobulins / Q000032:analysis / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping
D003432:Cross-Linking Reagents; D007136:Immunoglobulins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D020794:Receptor Protein-Tyrosine Kinases