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Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)
Author(s) -
Woo Sang B.,
Neet Kenneth E.,
Whalen Christopher
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070418
Subject(s) - tropomyosin receptor kinase a , nerve growth factor , neurotrophin , low affinity nerve growth factor receptor , chemistry , signal transduction , receptor , microbiology and biotechnology , neurite , biology , biochemistry , in vitro
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 pl40TrkA. NGF binding to pl40TrkA 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 p1 of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, K d , 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 × 10 5 M −1 s −1 and 1.6 × lO −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.