Molecular and biochemical characterization of the human trk proto-oncogene.

Abstract

Molecular analysis of the human irk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase gene family. This locus, which we now designate the trk proto-oncogene, codes for a protein of 790 amino acid residues that has several features characteristic of cell surface receptors. They include (i) a 32-amino-acid-long putative signal peptide, (ii) an amino-terminal moiety (residues 33 to 407) rich in consensus sites for N-glycosylation, (iii) a transmembrane domain, (iv) a kinase catalytic region highly related to that of other tyrosine kinases, and (v) a very short (15 residue) carboxy-terminal tail. Residues 1 to 392 were absent in the trk oncogene, as they were replaced by tropomyosin sequences. However, no other differences were found between the transforming and nontransforming trk alleles (residues 392 to 790), suggesting that no additional mutations are required to activate the transforming potential of this gene. The human trk proto-oncogene codes for a 140,000-dalton glycoprotein, designated gpl4QPrOtOt-k. However, its primary translational product is a 110,000-dalton glycoprotein which becomes inmediately glycosylated, presumably during its translocation into the endoplasmic reticulum. This molecule, designated gpll0Protofrk, is further glycosylated to yield the mature form, gpl40Prototrk. Both gpll0Proto-"k and gpl40PrOtO'-r proteins possess in vitro kinase activity specific for tyrosine residues. Finally, iodination of intact NIH 3T3 cells expressing trk proto-oncogene products indicated that only the mature form, gpj40Proto-frk, cross the plasma membrane, becoming exposed to the outside of the cell. These results indicate that the product of the human trk locus is a novel tyrosine kinase cell surface receptor for an as yet unknown ligand.

Specific TrkA survival signals interfere with different apoptotic pathways

“…The human TrkA cDNA was stably expressed in Rat1MycER TM cells using the expression vector pMEX-neo (Martin-Zanca et al, 1989). Expression of the 140 and 110 kDa TrkA proteins was monitored by immunoprecipitation with the TrkA-speci®c monoclonal antibody MGR12 (Cabrera et al, 1996) followed by Western blot analysis using a pan-Trk antibody (anti-203;Martin-Zanca et al, 1989).…”

“…cDNAs encoding the full length wt-TrkA receptor, or the single-site mutants (Y490F, Y785F) were expressed using the pMEX-neo expression vector (Martin-Zanca et al, 1989). The kinase-inactive mutant (K538A) was expressed from a pCMV5-based expression vector (a gift from Dr Moses Chao, Cornell University, NY), and the double mutant (Y490/785F) from a pLNCX-5-derived expression vector (a gift from Dr David Kaplan, McGill University, Montreal).…”

“…TrkA expression was analysed by immunoprecipitation using a monoclonal antibody speci®c for human TrkA (MGR12; Cabrera et al, 1996) followed by Western blotting using a pan-Trk antibody (anti-203) raised against a synthetic peptide corresponding to the 14 carboxy-terminal aminoacid residues in human TrkA (Martin-Zanca et al, 1989). NGF-induced TrkA phosphorylation was analysed after treating the cells with 100 ng/ml of NGF, at 378C, in serum-free medium.…”

Specific neurotrophin binding to leucine‐rich motif peptides of TrkA and TrkB

“…The rat TrkA [22] and rat TrkB [23] sequences correspond to the ones of the L2A and L2B peptides used in this work. The sequences of human TrkA, mouse TrkB, and chick TrkB were taken from Martin-Zanca et al [19], Klein et al [12], and Dechant et al [3], respectively. The localizations of the motifs within the respective proteins are indicated by the numbers on either side of the sequences.…”

Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

“…Previous reports have shown that expression of TrkA, TrkB or TrkC in NIH3T3 ®broblasts induces a potent mitogenic response when stimulated with their respective ligands (Cordon-Cardo et al, 1991; Glass et al, 1991;Lambelle et al, 1991). Indeed, TrkA was originally identi®ed as an oncogenic protein that led to the ligand-independent transformation of NIH3T3 cells (Martin-Zanca et al, 1989).…”