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Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains
M Takahashi1, Y Buma, T Iwamoto
1Laboratory of Experimental Pathology, Aichi Cancer Center Research Institute, Japan.
Abstract:
The nucleotide sequence of the ret proto-oncogene has been determined from cDNA clones isolated from a cDNA library of a THP-1 human monocytic leukemia cell line. Analysis of this sequence indicates that it encodes a protein which is structurally related to transmembrane receptors with a cytoplasmic tyrosine kinase domain. Unlike most growth factor receptors, it contains two hydrophobic regions which are potential transmembrane domains. Comparison of the sequence of the ret proto-oncogene with that of the ret transforming gene revealed that, in addition to the amino-terminal truncation, the last 51 carboxy-terminal amino acids of the ret proto-oncogene were replaced with 9 unrelated amino acids of the ret transforming gene. The focus forming activity of the ret cDNA, containing both amino-terminal and caboxy-terminal truncations, was approximately 13-fold higher than that of cDNA containing only the amino-terminal truncation. This suggests a possible role for the carboxy-terminal sequence in activation of transforming potential of the ret proto-oncogene. When transcription of the ret proto-oncogene was examined in a variety of mouse tissues, we detected its transcription in normal mouse spinal cord and lymphadenopathy of C3H/HeJ-gld/gld and MRL/Mp-lpr/lpr mice.
Insights
The ret proto-oncogene, a receptor tyrosine kinase, was sequenced. Its carboxy-terminal alterations significantly enhance its transforming potential, suggesting a role in oncogenesis.
Area of Science:
- Molecular Biology
- Oncogenesis
- Signal Transduction
Background:
- The ret proto-oncogene encodes a receptor tyrosine kinase involved in cell growth and differentiation.
- Alterations in proto-oncogenes can lead to the formation of oncogenes, driving cancer development.
- Understanding the structural and functional changes in ret is crucial for comprehending its role in oncogenesis.
Purpose of the Study:
- To determine the nucleotide sequence of the ret proto-oncogene.
- To analyze the structural relationship of the ret protein to other transmembrane receptors.
- To investigate the impact of carboxy-terminal modifications on the transforming activity of the ret gene.
Main Methods:
- Isolation of cDNA clones from a THP-1 human monocytic leukemia cell line library.
- Nucleotide sequencing of the ret proto-oncogene.
- Comparative sequence analysis between ret proto-oncogene and ret transforming gene.
- Functional analysis of ret cDNA constructs with varying truncations.
Main Results:
- The ret proto-oncogene sequence encodes a transmembrane receptor with a cytoplasmic tyrosine kinase domain and two potential transmembrane regions.
- The ret transforming gene differs from the proto-oncogene by N-terminal truncation and replacement of the C-terminal 51 amino acids with 9 unrelated residues.
- Ret cDNA with both N- and C-terminal truncations exhibited a 13-fold higher focus-forming activity compared to N-terminal truncation alone.
- Transcription of the ret proto-oncogene was detected in normal mouse spinal cord and in specific mouse models of lymphadenopathy.
Conclusions:
- The carboxy-terminal sequence of the ret proto-oncogene may play a role in its activation and transforming potential.
- Structural alterations, particularly C-terminal modifications, are critical for the oncogenic activity of the ret gene.
- The ret proto-oncogene is transcribed in the spinal cord, suggesting potential roles in neural development or function.