Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor

Abstract

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and C D change. During heating at 100 "C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.

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

“…As previously reported, the spectra of NGF consists primarily of contributions from /?-strand and random coil structure (Radziejewski et al, 1992;Timm & Neet, 1992;Narhi et al, 1993). The spectra of TrkA-RED has a characteristic contribution from a-helical structure in the 215-230 nm region and from /?-structure in the 195-205 nm region.…”

“…The high amount of /?-structure is consistent with two IgG-like domains in TrkA-RED, predicted from the sequence. The conformation of this extracellular domain is distinct from that of p75LANR, which is mainly random coil (Timm et al, 1992), but is similar to that of other receptor tyrosine kinases such as the epidermal growth factor (EGF) receptor and the insulin receptor (Greenfield et al, 1989;Schaefer et al, 1992).…”

“…We have previously shown a small change in conformation by CD measurements when each of the neurotrophins interacts with the common low-affinity receptor, p75LANR (Timm et at., 1992(Timm et at., , 1994. A similar experiment was conducted to determine if a conformational change occurred when TrkA-RED bound NGF.…”

“…DSS in DMSO was added to a final concentration of 150 p M and incubated for 30 min at room temperature to crosslink NGF to TrkA-RED. Samples were boiled in sample buffer and separated on a 12% SDS-PAGE and visualized either by autoradiography (Timm et al, 1992) or using a GS-363 Molecular Imager System (Bio-Rad). The relative band intensity was fit to a simple competition curve by a nonlinear least-squares fitting program to obtain an apparent EC50 (Fig.…”

The mature part of proNGF induces the structure of its pro-peptide

“…6B). The second transition probably reflects the unfolding of the mature part, as the highly homologous murine NGF had been shown to unfold in this concentration range [20]. The small plateau at very low GdmCl concentrations probably represents native proNGF species.…”