HSV-1 vector-mediated gene transfer of the human nerve growth factor receptor p75hNGFR defines high-affinity NGF binding

Abstract

A series of recombinant herpes simplex virus (HSV-1) vectors have been constructed that encode either the full-length cDNA of the human p75 NGF receptor (p75hNGFR) or truncated forms of the receptor. Infection of cultured fibroblast cells with viral stocks results in abundant expression of all three cDNAs, as detected by affinity cross-linking, immunoblot analysis, and equilibrium binding. Furthermore, viral infection of primary neuronal cultures gives easily detectable p75 expression by immunofluorescence and affinity cross-linking. When p75 was introduced by viral infection into fibroblast cells expressing the trk proto-oncogene, a new binding site was created, consistent with high-affinity NGF binding. This site is not created by the coexpression of truncated forms of p75 that lack either the extracellular ligand binding domain or the cytoplasmic domain of the receptor, suggesting that both of these regions of the receptor are required for the formation of the high-affinity NGF binding site. Hence, these HSV-1 vectors give rise to appropriate NGF receptor binding after viral infection. The application of these HSV-1 constructs to primary neuronal culture and in vivo models of p75NGFR function is discussed.

Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer's disease.

“…It was shown that in mature oligodendrocytes (10) NTR and p140 trkA are expressed on the cell surface, NGF receptor binding affinity is in the picomolar range (35), and NGF signals through p140 trkA leading to cell survival (36). Indeed, in the adult organism, presumptively through coordinated binding to both p75 NTR and p140 trkA , NGF can augment survival of aged neurons (37)(38)(39) as well as the survival of neurons after various injuries like hypoglycemia (40,41), axotomy (42)(43)(44), or exposure to neurotoxic substances (45).…”

Modeled structure of the 75-kDa neurotrophin receptor

“…NTR undergoes a change in affinity from a Kd of 3 nM to approximately 30 pM upon association with members of the trk tyrosine kinase family of NTRs, presumably by formation of a physical complex Battleman et al, 1993;Benedetti et al, 1993;Barker & Shooter, 1994;Hantzopoulos et al, 1994;Mahadeo et al, 1994;Wolf et al, 1995). The repeat unit 11 of NTR displays features that would indicate a functional role in intersubunit contacts.…”

“…Although not accepted by all investigators, NTR interacts with gpl40trk on both sides of the cell membrane to form the highaffinity binding complex (Hempstead et al, 1990;Battleman et al, 1993;Benedetti et al, 1993;Mahadeo et al, 1994;Wolf et al, 1995). Intracellular domain contacts appear to be essential for bringing the two different subunits into the complex, whereas their extracellular domains establish a high-affinity binding site (Wolf et al, 1995).…”

“…Association with gpl40trk subunits has been highly controversial despite evidence of participation by NTR in high-affinity complexes (Berg et al, 1991;Hempstead et al, 1991;Battleman et al, 1993;Benedetti et al, 1993;Barker & Shooter, 1994;Hantzopoulos et al, 1994;Mahadeo et al, 1994;Wolf et al, 1995). Failure of chemical crosslinking to reveal a physical complex between NTR and gpl40trk (Meakin & Shooter, 1991) and a report that gpl40trk homodimers account for NGF signaling (Jing et al, 1992) have suggested that NTR does not form a heterodimer with gpl4Otrk.…”

The Extracellular Domain of p75NTR Is Necessary to Inhibit Neurotrophin-3 Signaling through TrkA

“…In addition, p75 NTR regulates Trk receptor signaling. p75 NTR potentiates NGF activation of TrkA at low ligand concentrations (15,16) and collaborates with TrkA to form high affinity binding sites (17)(18)(19). Furthermore, the signaling pathways initiated by p75 NTR block Trk receptor signaling in certain contexts (20,21) and synergize with Trk receptor signaling in other contexts (22).…”