Isolation of a Novel β4 Integrin-binding Protein (p27BBP) Highly Expressed in Epithelial Cells*
- Stefano Biffo‡§,
- Francesca Sanvito‡¶,
- Silvana Costa‡,
- Laura Preve‡‖,
- Raffaella Pignatelli‡,
- Laura Spinardi‡‖ and
- Pier Carlo Marchisio‡**
- From the ‡Department of Biological and Technological Research (DIBIT), San Raffaele Scientific Institute, 20132 Milano, Italy, the **Department of Biomedical Sciences and Human Oncology, University of Torino, 10126 Torino, Italy, and the¶Department of Pathology, San Raffaele Scientific Institute, 20132 Milano, Italy
Abstract
The integrin β4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using β4 cytodomain uncovered a protein called p27BBP that represents a β4 interactor. Both in yeast and in vitro, p27BBP binds the two NH2-terminal fibronectin type III modules of β4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27BBP revealed that p27BBP 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, p27BBP mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27BBP COOH-terminal domain showed that all β4-containing epithelial cell lines expressed p27BBP. The p27BBP protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27BBP both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27BBP immunoreactivity was both nuclear and in the membrane closely apposed to β4. These results suggest that the p27BBP is an in vivointeractor of β4, possibly linking β4 to the intermediate filament cytoskeleton.
Footnotes
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↵* This work was supported by Telethon Grants 762 (to S. B.) and 826 (to P. C. M.) and also by Consiglio Nazionale delle Ricerche target project Applicazion: Cliniche della Ricerca Oncologica (ACRO) Associazione Italiana Ricerca sul Cancro (AIRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y11435 (human complete sequence of p27BBP) andY11460 (mouse partial sequence of p27BBP).
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↵§ To whom correspondence should be addressed: DIBIT, Dept. of Biological and Technological Research, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy. Tel.: 39-2-26434857; Fax: 39-2-26434855; E-mail: biffos{at}dibit.hsr.it.
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↵‖ Both authors supported by the Neuroscience Ph.D. program of the University of Torino.
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↵1 The abbreviations used are: PCR, polymerase chain reaction; FN, fibronectin; PBS, phosphate-buffered saline; BBP, β4-binding protein; GST, glutathioneS-transpherase; CLSM, confocal laser scanning microscopy.
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↵2 S. Biffo, unpublished observations.
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↵3 S. Biffo and F. Sanvito, unpublished observations.
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- Received April 14, 1997.
- Revision received September 5, 1997.
