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© The Rockefeller University Press, 0021-9525/1997//1333 $5.00
The Journal of Cell Biology, Volume 136, Number 6, , 1997 1333-1347

Article

The Localization of Bullous Pemphigoid Antigen 180 (BP180) in Hemidesmosomes Is Mediated by Its Cytoplasmic Domain and Seems to be Regulated by the β4 Integrin Subunit



Luca Borradori, Peter J. Koch*, Carien M. Niessen, Stefan Erkeland, Manuel R. van Leusden, and Arnoud Sonnenberg

Division of Cell Biology, The Netherlands Cancer Institute, NL-1066 CX Amsterdam; and * Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH2 terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH2 terminus of the cytoplasmic domain of BP180.

To investigate the effect of the {alpha}6β4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack {alpha}6β4 and BP180, with cDNAs for BP180 as well as for human {alpha}6A and β4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with {alpha}6β4. In contrast, when cells were transfected with cDNAs for {alpha}6A and mutant forms of β4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with {alpha}6β4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for {alpha}6A and a β4/β1 chimera, in which the cytoplasmic domain of β4 was replaced by that of the β1 integrin subunit, BP180 was not colocalized with the {alpha}6β4/β1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of β4 determine the subcellular distribution of BP180.

Abbreviations used in this paper: BP180, bullous pemphigoid antigen 180; BP230, bullous pemphigoid antigen 230; EB, epidermolysis bullosa; FNIII, type III fibronectin repeat; HD, hemidesmosomes; IF, intermediate filament; TAM, tyrosine activation motif.

We are indebted to Dr. K. B. Yancey, in whose laboratory at the Dermatology Branch, National Cancer Institute, National Institutes of Health (Bethesda, MD), part of the cloning of BP180 was performed during a postdoctoral fellowship (L. Borradori). We thank Drs. K. Owaribe, J. Kennel, J.R. Stanley, and J.C.R. Jones for kindly providing antibodies. We are grateful to Drs. E. Roos, J. Neefjes, P. Engelfriet, C. Gimond, and C. Baudoin for critical reading of the manuscript and to Dr. T. Sixma for helpful discussion about structural analysis predictions. Dr. L. Oomen is thanked for excellent assistance with the confocal laser microscope and N. Ong for photographic work.

Please address all correspondence to Arnoud Sonnenberg, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, NL1066 CX Amsterdam, The Netherlands.Tel: 31 20 5121942. Fax: 31 20 5121944.


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