The localization of bullous pemphigoid antigen 180 (BP180) in hemidesmosomes is mediated by its cytoplasmic domain and seems to be regulated by the beta 4 integrin subunit

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 6 beta 4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack alpha 6 beta 4 and BP180, with cDNAs for BP180 as well as for human alpha 6A and beta 4. 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 6 beta 4. In contrast, when cells were transfected with cDNAs for alpha 6A and mutant forms of beta 4, 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 6 beta 4, but remained diffusely distributed at the cell surface, Moreover, in cells transfected with cDNAs for alpha 6A and a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of the beta 1 integrin subunit, BP180 was not colocalized with the alpha 6 beta 4/beta 1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of beta 4 determine the subcellular distribution of BP180.