Characterization of Ret-Shc-Grb2 complex induced by GDNF, MEN 2A, and MEN 2B mutations.
We analyzed the intracellular signalling pathway through Ret activated by glial-cell-line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A and 2B mutations. The results showed that all of them induce a signal transducing complex consisting of Ret, Shc, and Grb2 proteins. In addition, GDNF clearly activated a Ras-MAPK pathway in human neuroblastoma cells. Rat is expressed mainly as two isoforms that differ in the carboxy-terminal sequence: a long isoform (1114 amino acids) and a short isoform (1072 amino acids). The long isoform contains the consensus sequence for binding of the Shc PTB domain but not of its SH2 domain, whereas the short isoform has the consensus sequences for binding of both domains. In vitro binding assay revealed that the long isoform of the MEN2A-Ret protein and both isoforms of the MEN2B-Ret protein bound preferentially to the Shc PTB domain. On the other hand, the short isoform of MEN2A-Ret bound to the PTB and SH2 domains. In neuroblastoma cells expressing both isoforms of Ret, its activation by GDNF also resulted in the binding of both domains. GDNF and MEN 2A mutations activate Ret by inducing its dimerization, whereas the MEN 2B mutation increases Ret catalytic activity without dimerization. Our results thus suggest that Ret dimerization might be required for binding of the Shc SH2 domain to the short isoform.
Ohiwa
M
M
Department of Pathology, Nagoya University School of Medicine, Japan.
Murakami
H
H
Iwashita
T
T
Asai
N
N
Iwata
Y
Y
Imai
T
T
Funahashi
H
H
Takagi
H
H
Takahashi
M
M
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D016384:Consensus Sequence; D029721:Drosophila Proteins; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D011905:Genes, ras; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006801:Humans; D051379:Mice; D018813:Multiple Endocrine Neoplasia Type 2a / Q000235:genetics*; D018814:Multiple Endocrine Neoplasia Type 2b / Q000235:genetics*; D009154:Mutation; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D009447:Neuroblastoma; D017354:Point Mutation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D018631:ras Proteins / Q000302:isolation & purification / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D029721:Drosophila Proteins; C495039:GDNF protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495040:Gdnf protein, mouse; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; C495001:Ret protein, rat; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins