The survival and functional maintenance of vertebrate neurons critically depends on the availability of specific neurotrophic factors. So far, only two such factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been characterized and shown to have the typical features of secretory proteins. This characterization has been possible because of the extraordinarily large quantities of NGF in some adult tissues, and the virtually unlimited availability of brain tissue from which BDNF was isolated. Both NGF and BDNF promote the survival of distinct neuronal populations in vivo and are related in their primary structure, suggesting that they are members of a gene family. Although there is little doubt about the existence of other such proteins, their low abundance has rendered their identification and characterization difficult. Taking advantage of sequence identities between NGF and BDNF, we have now identified a third member of this family, which we name neurotrophin-3. Both the tissue distribution of the messenger RNA and the neuronal specificity of this secretory protein differ from those of NGF and BDNF. Alignment of the sequences of the three proteins reveals a remarkable number of amino acid identities, including all cysteine residues. This alignment also delineates four variable domains, each of 7-11 amino acids, indicating structural elements presumably involved in the neuronal specificity of these proteins.

Immortalized cell lines have been generated from embryonic mouse neuroepithelium by infection with a retrovirus containing the c-myc oncogene. The morphology and the antigenic phenotype of the cloned cell lines are characteristic of normal neuroepithelium. Although the cell lines are stable and do not spontaneously differentiate, morphological changes can be induced with both acidic and basic fibroblast growth factor. Fibroblast growth factor at S ng/ml stimulates differentiation of the neuroepithelial cells, and it has been shown that the cloned cell line 2.3D can differentiate into astrocytes, containing glial fibrillary acidic protein, and neurons, expressing the A2B5 marker and neurofilaments. This indicates that some cells in the neuroepithelium at embryonic day 10 are multipotent and are not restricted to either the glial or neuronal cell lineage. The cell lines also can be induced with interferon y to express class I and class II histocompatibility antigens. The response of the c-mycimmortalized cell lines to these two factors is similar to that observed with freshly isolated neuroepithelium and suggests that such immortalized precursor populations are representative of the cells found in the developing neuroepithelium.

The specific interaction between a ligand and a protein is a key component in minimizing off-target effects in drug discovery. Investigating these interactions with membrane protein receptors can be quite challenging. In this report, we show how spectral variance observed in surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) can be correlated with ligand specificity in affinity-based assays. Variations in the enhanced Raman spectra of three peptide ligands (i.e., cyclic-RGDFC, cyclic-isoDGRFC, and CisoDGRC), which have different binding affinity to αvβ3 integrin, are reported from isolated proteins and from receptors in intact cancer cell membranes. The SERS signal from the purified proteins provides basis spectra to analyze the signals in cells. Differences in the spectral variance within the SERS and TERS data for each ligand indicate larger variance for nonspecific ligand–receptor interactions. The SERS and TERS results are correlated with single particle tracking experiments of the ligand-functionalized nanoparticles with purified receptors on glass surfaces and living cells. These results demonstrate the ability to elucidate protein–ligand recognition using the observed vibrational spectra and provide perspective on binding specificity for small-molecule ligands in intact cell membranes, demonstrating a new approach for investigating drug specificity.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are known to exist in solution as non-covalently linked homodimeric proteins. The recent elucidation of the crystal structure of the NGF homodimer, as well as the conservation of structural motifs in the neurotrophins, raised the possibility that neurotrophin heterodimers might also occur. The formation of BDNFNT-3 heterodimers was explored using a vaccinia virus expression system. Upon co-infection of cells with viruses expressing BDNF and NT-3, we could identify a BDNFNT-3 heterodimer as a biosynthetic product and separate it from the BDNF and NT-3 homodimers. We could also show that the BDNF/NT-3 heterodimers can be formed irrespective of wild-type or exchanged prosequences, indicating that prosequence specificity does not influence dimer formation. In all neuronal survival assays that were used, the BDNFNT-3 heterodimer was shown to be 10-fold less active compared with a mixture of BDNF and NT-3 homodimers. This lower specific activity was also measured in a neuronal population co-expressing receptors for BDNF and NT-3. The low biological activity of the heterodimer observed with neurons was not paralleled by a reduced ability of the heterodimer to interact with trkB or trkC receptors, as assessed by the induction of tyrosine phosphorylation of these receptors expressed by fibroblast cell lines.

No abstract