Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF).
Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and p140TrkA. NGF binding to p140TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a pI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 x 10(5) M(-1) s(-1) and 1.6 x 10(-3) s(-1), respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.
Woo
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Department of Biological Chemistry, Finch University of Health Sciences/The Chicago Medical School, Illinois 60064, USA.
Whalen
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Neet
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D000818:Animals; D015374:Biosensing Techniques; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D006031:Glycosylation; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D008968:Molecular Conformation; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry*; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D003432:Cross-Linking Reagents; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA