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Deletion mutant of FGFR4 induces onion-like membrane structures in the nucleus
Vigdis Sørensen1, Andreas Brech, Denis Khnykin
1Institute for Cancer Research, The Norwegian Radium Hospital, Department of Biochemistry, Montebello, 0310 Oslo, Norway.
Abstract:
The expression of several deletion mutants of fibroblast growth factor receptor 4 (FGFR4) was studied in COS-1 cells. FGFR4-mutants lacking most of the extracellular region did not efficiently reach the plasma membrane but accumulated in the endoplasmic reticulum (ER) and Golgi body. A mutant FGFR4 lacking the kinase domain as well as most of the extracellular region (DeltaExt/R4Tth) had a distinct intracellular distribution. It localized in part to the nucleus, where it exhibited a striking spotted pattern. Ultrastructural studies showed that the nuclear spots consisted of several layers of membrane that were folded into onion-like structures at the nucleoplasmic side of the nuclear envelope. These intranuclear structures did not contain nuclear pores but were positive for the ER proteins calreticulin and protein disulfide isomerase, in addition to abundant DeltaExt/R4Tth. Formation of the intranuclear structures was sensitive to inhibition of protein kinase C. Live microscopy of a green-fluorescent-protein/DeltaExt/R4Tth fusion protein showed that the intranuclear structures were stable and immobile, suggesting that they function as deposits of the overexpressed mutant and associated membrane. The DeltaExt/R4Tth protein also induced formation of densely packed membrane stacks in the cytosol and we suggest a model were the intranuclear structures are formed by invagination of ER-derived membrane stacks into the nucleus.
Insights
Fibroblast growth factor receptor 4 (FGFR4) mutants accumulate in the endoplasmic reticulum. A specific mutant, DeltaExt/R4Tth, forms unique, stable, onion-like membrane structures within the nucleus.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Biochemistry
Background:
- Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane receptor tyrosine kinase.
- Proper localization and function of receptor tyrosine kinases are crucial for cellular signaling.
- Aberrant FGFR4 expression and localization are implicated in various diseases.
Purpose of the Study:
- To investigate the intracellular localization and behavior of deletion mutants of FGFR4.
- To characterize the unique nuclear localization and structure of a specific FGFR4 mutant (DeltaExt/R4Tth).
- To elucidate the mechanism of formation for these novel intranuclear structures.
Main Methods:
- Expression of FGFR4 deletion mutants in COS-1 cells.
- Immunofluorescence microscopy and ultrastructural analysis (electron microscopy).
- Live-cell imaging using green fluorescent protein (GFP) fusion proteins.
- Biochemical assays to assess protein localization and sensitivity to inhibitors.
Main Results:
- FGFR4 mutants lacking significant extracellular regions accumulated in the endoplasmic reticulum (ER) and Golgi.
- A specific mutant, DeltaExt/R4Tth (lacking extracellular and kinase domains), localized to the nucleus in a distinct spotted pattern.
- Ultrastructural studies revealed these nuclear spots as onion-like membrane structures positive for ER proteins.
- Formation of these intranuclear structures was sensitive to protein kinase C inhibition.
- Live microscopy demonstrated the stability and immobility of these structures, suggesting they act as deposits.
Conclusions:
- Overexpressed FGFR4 mutants exhibit aberrant intracellular trafficking.
- The DeltaExt/R4Tth mutant forms unique, stable intranuclear membrane structures derived from the ER.
- These structures may represent a novel mechanism for sequestering overexpressed proteins and associated membranes.
- The findings provide insights into protein mislocalization and organelle dynamics in response to overexpression.
