Isolation of a novel beta4 integrin-binding protein (p27(BBP)) highly expressed in epithelial cells
1Department of Biological and Technological Research (DIBIT), San Raffaele Scientific Institute, 20132 Milano, Italy. biffo@dibit.hsr.it
Insights
A novel protein, p27(BBP), interacts with integrin beta4 (β4) and may link it to intermediate filaments. This interaction is crucial for hemidesmosome formation and cellular signaling in epithelial cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Integrin beta4 (β4) possesses a lengthy cytodomain essential for hemidesmosome assembly.
- Hemidesmosomes are critical adhesive structures in epithelial cells, mediating cell-matrix interactions.
Purpose of the Study:
- To identify novel interactors of the integrin beta4 (β4) cytodomain.
- To characterize the function and localization of a newly discovered β4-interacting protein, p27(BBP).
Main Methods:
- Yeast two-hybrid screening to identify β4 interactors.
- In vitro binding assays and protein sequence analysis.
- Northern analysis, in situ hybridization, antibody generation, and subcellular fractionation.
- Confocal microscopy to determine protein localization.
Main Results:
- A previously unknown protein, p27(BBP), was identified as a β4 interactor, binding to its fibronectin type III modules.
- p27(BBP) mRNA is highly expressed in epithelia and proliferating embryonic cells.
- p27(BBP) localizes to the cytoplasm and nucleus, and partially co-localizes with β4 at the membrane, associating with intermediate filaments.
Conclusions:
- p27(BBP) is an in vivo interactor of integrin beta4 (β4).
- p27(BBP) may function as a linker between β4 and the intermediate filament cytoskeleton.
- This interaction is potentially significant for cellular signaling and hemidesmosome formation.
Abstract:
The integrin beta4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using beta4 cytodomain uncovered a protein called p27(BBP) that represents a beta4 interactor. Both in yeast and in vitro, p27(BBP) binds the two NH2-terminal fibronectin type III modules of beta4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27(BBP) revealed that p27(BBP) was not previously known and has no homology with any isolated mammalian protein, but 85% identical to a yeast gene product of unknown function. Expression studies by Northern analysis and in situ hybridization showed that, in vivo, p27(BBP) mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27(BBP) COOH-terminal domain showed that all beta4-containing epithelial cell lines expressed p27(BBP). The p27(BBP) protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27(BBP) both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27(BBP) immunoreactivity was both nuclear and in the membrane closely apposed to beta4. These results suggest that the p27(BBP) is an in vivo interactor of beta4, possibly linking beta4 to the intermediate filament cytoskeleton.
