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Identification of a novel activated form of the keratinocyte growth factor receptor by expression cloning from
K Sakaguchi1, M V Lorenzi, H Matsushita
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Building 37 Room 1E24, Bethesda, Maryland, MD 20892-4255, USA.
Abstract:
Parathyroid adenomas are benign tumors in the parathyroid glands, whose pathogenesis is largely unknown. We utilized an expression cDNA cloning strategy to identify oncogenes activated in parathyroid adenomas. An expression cDNA library was prepared directly from a clinical sample of parathyroid adenoma tissue, transfected into NIH3T3 cells, and foci of morphologically transformed cells were isolated. Following plasmid rescue, we identified cDNAs for the keratinocyte growth factor receptor at a high frequency. Interestingly, approximately half of the clones encoded a variant receptor containing an altered C-terminus. Analysis of the transforming activity of the variant receptor revealed that the altered C-terminus up-regulated the transforming activity in a ligand-independent manner. The higher transforming activity was not accompanied by increase of dimerization or overall autophosphorylation of the receptor. However, tyrosine phosphorylation of downstream receptor substrates, including Shc isoforms and possibly FRS2, are increased in the transfectants expressing the parathyroid tumor-derived receptor. Genomic analysis showed that a previously unidentified exon was used to form the novel isoform. This alternative splicing appears to occur preferentially in parathyroid adenomas.
Insights
Researchers identified a variant keratinocyte growth factor receptor (KGFR) in parathyroid adenomas. This altered receptor promotes tumor growth independently of its usual signaling pathway.
Area of Science:
- Oncology
- Molecular Biology
- Endocrinology
Background:
- Parathyroid adenomas are benign tumors of the parathyroid glands.
- The underlying molecular mechanisms driving parathyroid adenoma development remain largely unknown.
- Identifying activated oncogenes is crucial for understanding tumor pathogenesis.
Purpose of the Study:
- To identify activated oncogenes in parathyroid adenomas using an expression cDNA cloning strategy.
- To characterize the functional role of identified genes in tumor development.
- To investigate the specific alterations and transforming activity of identified receptors.
Main Methods:
- Construction of an expression cDNA library directly from parathyroid adenoma tissue.
- Transfection of the cDNA library into NIH3T3 cells to isolate morphologically transformed foci.
- Plasmid rescue and sequencing to identify cloned cDNAs.
- Analysis of variant receptor transforming activity, dimerization, autophosphorylation, and downstream substrate phosphorylation.
- Genomic analysis to identify novel exons and alternative splicing events.
Main Results:
- The keratinocyte growth factor receptor (KGFR) was identified at a high frequency in parathyroid adenomas.
- Approximately half of the identified KGFR clones encoded a variant receptor with an altered C-terminus.
- The variant KGFR exhibited increased transforming activity in a ligand-independent manner.
- Increased tyrosine phosphorylation of downstream substrates like Shc isoforms and FRS2 was observed.
- Genomic analysis revealed a novel exon utilized in the variant KGFR, suggesting alternative splicing specific to parathyroid adenomas.
Conclusions:
- A variant KGFR with an altered C-terminus is preferentially expressed in parathyroid adenomas.
- This variant KGFR possesses enhanced transforming activity, contributing to tumor development.
- Alternative splicing leading to this novel KGFR isoform is a potential mechanism in parathyroid tumorigenesis.