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Identification of a point mutation in the folate receptor gene that confers a dominant negative phenotype
1Department of Pediatrics and Pharmacology, University of Texas Southwestern Medical Center at Dallas 75235-9063.
Abstract:
UM-SCC-38 cells, a squamous cell carcinoma cell line of the head and neck, express limited amounts of folate receptor alpha antigen which is not capable of binding either folic acid or 5-methyltetrahydrofolic acid. Three distinct mutations in the open reading frame of the folate receptor were identified. We now show that the three mutants are nonfunctional with respect to folic acid binding because the protein products do not bind folate. Additionally, a study of MA104 cells (a receptor-positive cell line) transfected with each mutant was done. Expression of one mutant, FR-67, results in a dominant negative phenotype because folate binding is significantly reduced although membrane antigen is significantly increased. Coexpression of FR-67 and the normal protein in MA104 cells also results in large, bright clusters of receptor protein inside the cell around the nucleus when visualized using indirect immunofluorescence. These clusters are not found in cells that express either normal or FR-67 protein alone. In conclusion, this study provides the first evidence of a mutant folate receptor protein capable of affecting normal receptor function in a dominant negative manner.
Insights
Mutant folate receptors in head and neck cancer cells are nonfunctional and can block normal folate receptor alpha function. This dominant negative effect impacts folate binding and cellular localization.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Head and neck squamous cell carcinoma (HNSCC) cells, like UM-SCC-38, exhibit limited folate receptor alpha (FRα) expression.
- This limited FRα expression is insufficient for binding folic acid or 5-methyltetrahydrofolic acid.
Purpose of the Study:
- To investigate the functional consequences of identified mutations in the folate receptor alpha open reading frame.
- To determine if mutant folate receptor proteins can interfere with normal folate receptor function.
Main Methods:
- Analysis of three distinct folate receptor alpha mutations within the open reading frame.
- Functional assays to assess folate binding capabilities of mutant proteins.
- Transfection of MA104 cells (a receptor-positive cell line) with mutant folate receptor genes.
- Immunofluorescence microscopy to visualize protein expression and localization.
Main Results:
- The three identified folate receptor alpha mutants are nonfunctional, failing to bind folate.
- Expression of the FR-67 mutant in MA104 cells leads to a dominant negative phenotype, characterized by reduced folate binding and increased membrane antigen levels.
- Coexpression of FR-67 with normal folate receptor alpha in MA104 cells induces the formation of intracellular protein clusters near the nucleus.
Conclusions:
- This study presents the first evidence of a mutant folate receptor alpha protein exhibiting a dominant negative effect on normal receptor function.
- The FR-67 mutant can disrupt normal folate receptor alpha activity and cellular localization.
- Understanding these mutant receptor behaviors is crucial for potential therapeutic strategies in HNSCC.