180-kD bullous pemphigoid antigen type XVII collagen: Tissue-specific expression and molecular interactions with keratin 18

The 180-kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmemb rane protein, also known as type XVII collagen. In this study, potential in teractions of BPAG2 with other proteins expressed in epidermal keratinocyte s were explored by yeast two-hybrid system using the amino-terminal intrace llular domain of BPAG2 as a bait. Several independent interacting clones en coding keratin 18 (K18) were identified when the keratinocyte cDNA library cloned into the yeast two-hybrid activation domain vector, was screened. Th e peptide sequence responsible for the interaction of BPAG2 was restricted to amino acids 15-25, and substitution of a valine residue in the middle of this sequence by a proline (V23P) by site-directed mutagenesis abolished t he interaction. Further examination of the K18 sequences by restricted cDNA constructs in yeast two-hybrid system identified a carboxyl-terminal segme nt corresponding to helix 2B domain as critical for BPAG2 binding. The inte raction of BPAG2/K18 was confirmed by an in vitro protein-protein interacti on assay, which also confirmed that normal human keratinocytes express K18 in culture. The tissue specific expression of BPAG2 was first examined usin g a multi-tissue RNA blot. Human multiple tissue cDNA panels representing a variety of adult and fetal tissues as well as tumor cells were used as PCR -templates to study the expression patterns of both BPAG2 and K18. The resu lts demonstrated significant level of expression of BPAG2, besides in epide rmal keratinocytes, also in a variety of tissues with predominant epithelia l component, such as mammary, salivary and thyroid glands, colon, prostate, testis, placenta, and adult and fetal thymus, as well as in colon, pancrea tic and prostatic adenocarcinoma cell lines, and an ovarian carcinoma. As e xpected, K18 transcript is present in liver, pancreas, colon, placenta, and in fetal kidney. Collectively, the results suggest that BPAG2 has a relati vely bread tissue distribution including specialized and simple epithelia, and that within the tissues such as colon and placenta, BPAG2 may have dire ct interactions with K18, a keratin characteristically expressed in a simpl e epithelia. (C) 1999 Wiley-Liss, Inc.