Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)

Abstract

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 pl4OTrkA. NGF binding to pl4OTrkA 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 lo5 M" s -' and 1.6 X s", respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.Keywords: mutant; nerve growth factor; neurotrophin; receptor binding; receptor extracellular domain; recombinant; structure The growth factor (NGF) and its homologous neurotrophic factors are members of the growing family of neurotrophins that play a profound pharmacological role in development and survival of neuronal cells (Snider, 1994). Five members of the neurotrophin family have thus far been isolated, with NGF as the prototype for this family. The other members include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 ~ istry, Finch UHS/Chicago Medical School, 3333 Green Bay Rd., North Reprint requests to: Kenneth E. Neet, Department of Biological ChemChicago, Illinois 60064; e-mail: neetk@mis.finchcms.edu.Abbreviations; BDNF, brain-derived neurotrophic factor; CM5, carboxymethylated dextran matrix; DSS, disuccinimidyl suberate; EDC, N-ethyl-N'-(dimethylaminopropyI)carbodiimide; HBS, HEPES buffered saline; LANR, low-affinity neurotrophin receptor; NGF, nerve growth factor p subunit; NT3, ueurotrophin-3; NHS, N-hydroxysuccinimide; NT4/5, neurotrophin-4/5; PC12, pheochromocytoma 12 cell line; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sf21, Spodoptera frugiperda 21 (insect cell line); TrkA-RED, TrkA receptor extracellular domain (NT4/5); fish neurotrophin-6 (NT6) is the most newly discovered neurotrophin (Ip...

TrkA Receptor “Hot Spots” for Binding of NT-3 as a Heterologous Ligand

“…This rigidity seems quite unusual because generally ligand conformation is affected upon binding and NGF undergoes conformational changes upon binding to p75-ECD (33). Along these lines, CD spectra of TrkA-ECD in complex with NGF showed small but significant conformational changes in secondary structure (24).…”

“…Mouse NGF30 mAb is an anti-NGF mAb that when bound to NGF still allows high affinity binding of NGF to TrkA. The interaction of NGF-NGF30 complexes with TrkA affords survival but not differentiation of TrkA-expressing cells (23 (23,24). The Ig-C1(D4) and Ig-C2(D5) subdomains of human TrkA (TRKA-D4 -D5) were produced in yeast.…”

“…The integrity and frame of the constructs were verified by sequencing (data not shown). The human TrkA-ECD or rat chimeric ECD were each overexpressed in Sf21 insect cells in XL-401 medium (JRH Biosciences, Lenexa, KA) as described previously (24). Proteins were purified with their C-terminal His 6 tag by nickel-nitrilotriacetic acid chromatography and subsequent steps to near homogeneity as shown with SDS-polyacrylamide gels stained with Coomassie Blue (24).…”

“…The human TrkA-ECD or rat chimeric ECD were each overexpressed in Sf21 insect cells in XL-401 medium (JRH Biosciences, Lenexa, KA) as described previously (24). Proteins were purified with their C-terminal His 6 tag by nickel-nitrilotriacetic acid chromatography and subsequent steps to near homogeneity as shown with SDS-polyacrylamide gels stained with Coomassie Blue (24). 5 The human Trk-ECD wild type or rat 4.1 chimera was immobilized on a CM4 sensor chip using EDC/NHS coupling chemistry and analyzed on a Biacore 3000 instrument (Biacore, Piscataway, NJ) using BIA Evaluation software (version 4.0.1, Biacore).…”

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

“…Surface Plasmon Resonance (SPR) Spectroscopy-The TrkA ECD was prepared at 5 g/ml in 10 mM sodium acetate coupling buffer, pH 4.5, and immobilized to a CM4 sensor chip using N-(3-dimethylaminopropyl)-NЈ-ethylcarbodiimide/Nhydroxysuccinimide coupling chemistry on a Biacore 3000 instrument (Biacore, Piscataway, NJ) as described previously (69). All reagents were automatically introduced over the sensor chip in 10 mM HEPES, pH 7.4, 0.15 M NaCl, and 0.005% v/v surfactant P20 (HBS-P) at a flow rate of 30 l/min with a blank chip subtracted as control for nonspecific surface binding.…”