C C Drinkwater

Two distinct nerve growth factor receptor (NGFR) complexes are present on NGF-responsive cell types; these correspond to 100 kDa and 158 kDa for the fast (fNGFR) and the slow (sNGFR) NGFRs, respectively. Previous studies indicate that each complex is derived from a separate gene product and that the sNGFR contains tyrosine kinase activity. The cDNA encoding the fNGFR has previously been cloned. In this report, a rat trk protooncogene cDNA has been isolated from PC12 cells and Trk has been shown to bind NGF, generating a complex of 158 kDa. Characterization of NGF-Trk interactions indicates that Trk and NGF dissociate more slowly than do NGF and the fNGFR. Moreover, NGF-bound Trk is not destroyed by trypsin digestion whereas the NGF-fNGFR complex is sensitive to trypsin digestion. These observations suggest that the trk protooncogene product, expressed in the absence of the fNGFR, binds NGF with properties characteristic of the sNGFR, which was identified as the high-affinity NGFR on primary neurons and PC12 cells.

Mouse glandular kallikreins are encoded by a family of closely linked genes which are located on chromosome 7 at a site corresponding to the genetically defined Tam-l, Prt-4, and Prt-6 loci.We have characterized 24 kallikrein genes by genomic cloning and restriction mapping of 310 kilobase pairs of BALBfc mouse DNA.Most of these genes are highly homologous, have the same exonfintron organization, and are linked in clusters of up to 11 genes.Partial sequence analysis of the kallikrein genes has facilitated identification of those members of the family for which protein sequence data exist and assignment of those which are pseudogenes or encode proteins of unknown function.We find that a maximum of 14 mouse kallikrein genes have the potential to encode functional proteins.

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.

A series of mouse nerve growth factor (NGF) deletion mutants have been constructed using in vitro mutagenesis to define domains of the protein essential for its activity. Deletions of the amino or carboxyl termini of mature NGF or of an internal domain, which normally produces a surface-exposed reverse turn, have been analyzed. Mutants with deletions in the amino terminus or in the reverse turn retain significant biological activity, whereas, in contrast, a mutant NGF lacking the seven most carboxyl-terminal amino acids is appropriately synthesized but shows no measurable biological activity. These results suggests that the flexible carboxyl tail of NGF, and perhaps other neurotrophins, plays a crucial role in mediating receptor recognition and/or ligand binding.