QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 4 December 2002
doi: 10.1242/jcs.00241

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Koster, J. Articles by Sonnenberg, A. Search for Related Content PubMed PubMed Citation Articles by Koster, J. Articles by Sonnenberg, A.

Analysis of the interactions between BP180, BP230, plectin and the integrin 6ß4 important for hemidesmosome assembly

Jan Koster¹, Dirk Geerts^1,*, Bertrand Favre², Luca Borradori² and Arnoud Sonnenberg^1,

¹ The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
² University Hospital Geneva, Department of Dermatology, CH-1211 Geneva, Switzerland
^* Present address: Department of Human Genetics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam

View larger version (114K): [in a new window]   Fig. 1. Immunofluorescence analysis of hemidesmosomal components in PA-JEB/ß4 cells. PA-JEB/ß4 cells were fixed and immunolabeled for laminin-5 (A) or the hemidesmosomal markers ß4 (A,B,D,F), plectin (B,G,I), BP180 (C,E,F) or BP230 (C,H,I). Merged images (A,B,C,F,I) show laminin-5, plectin and BP180 in green, and ß4 and BP230 in red; co-localized staining appears in yellow. Note that ß4 and plectin (B), as well as BP230 and BP180 (C), are nearly completely co-localized, whereas ß4 and BP180 (F) or plectin and BP230 (I) show only partial co-localization. Bar, 10 µm.

View larger version (121K): [in a new window]   Fig. 2. Immunofluorescence analysis of hemidesmosomal components in GABEB cells. GABEB cells were fixed and immunolabeled for plectin (A), BP180 (B), BP230 (C) and ß4 (A-C). In D-I, GABEB cells were transfected with cDNA encoding wild-type BP180 tagged at the N-terminus with a FLAG-epitope (yellow square in diagrams at the bottom of D and H; diagrams represent cDNA encoding wild-type BP180) stained for BP180 (D,F,H,I), ß4 (E,F) or BP230 (G,I). Merged images (A-C,F) show plectin, BP180 and BP230 in green and ß4 in red. In the merged image I, BP180 is in green and BP230 is in red. Note that on reconstitution of BP180, BP230 is recruited into HD-like structures, whereas it is not recruited when BP180 expression is absent. Bars, 10 µm.

View larger version (15K): [in a new window]   Fig. 3. Yeast two-hybrid analysis of interactions between BP180 and BP230 and BP180 and ß4. (Top) Schematic representation of BP180. DSR, direct sequence repeat; TM, transmembrane region. (Bottom) Cotransformation of yeast host strain PJ69-4A with BP2301-555 or ß41115-1666 cDNA constructs fused to the Gal4 (AD) domain (in pACT2) and cDNA constructs encoding various fragments of BP180 fused to Gal4 (BD) domain (in pAS2.1) as indicated. Transformation mixtures were spread on SC-LT and SC-LTHA plates and grown at 30°C. Plating efficiency on selective SC-LTHA plates is expressed as a percentage of the plating efficiency on non-selective SC-LT plates from the same transformation. Plates were scored after 6 and 10 days. All efficiencies listed represent an average of multiple independent transformations. ++, plating efficiency on SC-LTHA is 80% of the plating on SC-LT, colonies are fully developed on day 5; +, 40-80% of the plating on SC-LT, small and large colonies on day 5; ±, 50% of the plating on SC-LT at 10 days of growth; -, no colonies on selective plates after 10 days of growth. Note that the interaction between BP180 and BP2301-555 requires a fragment of BP180 containing amino acids 145-230, whereas for the interaction of BP180 with ß41115-1666 other sequences are required. Identical results were obtained when, instead of BP2301-555, BP2301-1156 was used for assessment of the interactions with the different BP180 mutants.

View larger version (111K): [in a new window]   Fig. 4. Effects of expression of BP180 mutants on the localization of BP230 in GABEB keratinocytes. GABEB keratinocytes were transfected with cDNA encoding BP180145-230 (A,B) or BP1801-36 (C,D) and immunolabeled for BP180 (A-D), ß4 (A,C) or BP230 (B,D). In the merged images (A,C), BP180 is in green and ß4 in red, whereas in B and D, BP180 is in red and BP230 is in green. Staining for co-localization is in yellow. Diagrams at the bottom of panels A-D represent the various BP180 mutants. Note that deletion of amino acids 145-230 from BP180 does not abrogate the ability of BP180 to be recruited into HDs, whereas the recruitment of BP230 is impaired. Deletion of the N-terminal 36 residues of BP180 has no effect on the recruitment of either BP180 or BP230 into HDs. Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 5. Interaction between BP230 and BP180 involves the conserved Y subdomain of plakins and mapping of the interaction site on BP230 for ß4. The PJ69-4A yeast strain was co-transformed with a pAS2.1- or pPACT2-derived vector encoding BP1801-401 or ß41115-1666 and the corresponding complementary vectors encoding various fragments of BP230, desmoplakin or plectin as indicated. Two-hybrid interactions were analyzed by growth on selective SC-LTHA plates. (A) Top, schematic representation of BP230. The subdomains NN, Z, Y, X, W and V are those described by Green et al. (Green et al., 1992). These subdomains as well as the homologous repeat sequences designated B and C are indicated by boxes, shaded from white to gray. The box representing the rod domain has been shaded more darkly. Bottom, the interaction of BP1801-401 with the various fragments of BP230 could be shown independently of whether the cDNA constructs were cloned into the pAS2.1 or the pACT2 vector. Because of an autonomous transactivation of pAS2.1-ß41115-1666, the BP230 binding activity of ß41115-1666 could only be determined with the combination of pACT2-ß41115-1666 and pAS2.1-BP230. (B) Interactions of BP1801-401 with the Z-Y and Y domains of BP230, plectin and desmoplakin (DP) were only revealed when the cDNAs encoding the various domains of BP230, plectin and desmoplakin were inserted into pACT2 and the cDNA for BP1801-401 was inserted into pAS2.1. (C) The ß-galactosidase activity of the yeast transformants expressing BP1801-401 and the indicated BP230, plectin and desmoplakin constructs was quantified in a liquid culture assay using O-nitrophenyl ß-D-galactopyranoside as substrate. The negative interaction controls are pAS2.1-BP1801-401/pACT2 (2.9±0.3 ß-galactosidase units) and pAS2.1/pACT2 (2.4±0.2 ß-galactosidase units) and the positive controls (not shown) are p53/pSV-40 large T (77.3±10.1 ß-galactosidase units) and the complete Gal4 transcription factor in pCL1 (266.8±16.3 ß-galactosidase units). (D) Interactions between BP230 mutants and ß41115-1666 were assayed with the BP230 mutants fused to the Gal4 (BD) in pAS2.1 and ß41115-1666 fused to the Gal4 (AD) in pACT2. For further details, see Fig. 3. In panel D, the amino acids GGG, GSG and G correspond to the linker sequences placed in between the Gal4 (AD)- and the BP230-specific sequences. In B and D note that the interaction between BP230 and the cytodomain of BP180 only requires the Y domain of BP230, whereas for the interaction with ß4 the 56-most N-terminal residues of BP230 are involved. Panel E shows an alignment of the Z-Y regions in the different plakin proteins.

View larger version (88K): [in a new window]   Fig. 6. Expression of BP230 mutants in PA-JEB/ß4 keratinocytes. PA-JEB/ß4 cells were transfected with cDNAs encoding BP2301-887 (A,B), BP2301-2161 (C,E) or BP2301-2161,ZY (D,F), tagged at the N-terminus with an HA epitope (red circle). Cells were fixed and immunolabeled for BP180 (A-D), the HA-tagged BP230 mutants (A-F) and total BP230 (endogenous and transfected BP230) (E,F). In the merged images (A-D), BP180 is in green and the HA-tagged mutants are in red, whereas in (E,F), the HA-tagged BP230 mutant is in green and total BP230 is in red. Co-localized staining appeared in yellow. Diagrams at the bottom of panels A-F represent the various BP230 mutants. Note that the cell depicted in B represents a rare event, whereas the majority of the cells resemble those depicted in A. Also note that removal of the Z-Y domains results in the loss of BP230 recruitment into HD-like structures. Bars, 10 µm.

View larger version (18K): [in a new window]   Fig. 7. Yeast two-hybrid analysis of interactions between ß4 and BP230 and ß4 and BP180. (Top) Schematic representation of the cytoplasmic domain of ß4. FNII repeats are represented by boxes in which the number of the repeats is shown. TM, transmembrane region. (Bottom) Cotransformation of yeast host strain PJ69-4A with ß4 mutants in the pACT2 (AD) vector and BP2301-1156 or BP1801-401 in the pAS2.1 (BD) vector. For further details see Fig. 3. Note that BP230 requires the third and fourth FNIII repeats, as well as the last 20 amino acids of the CS of ß4, to allow an interaction, whereas BP180 only requires the third FNIII repeat.

View larger version (76K): [in a new window]   Fig. 8. Binding of ß4 to plectin is essential for the efficient recruitment of BP180 and BP230 into HDs. PA-JEB keratinocytes stably expressing a ß4 mutant that is unable to bind to plectin (ß4R1281W) were immunolabeled for ß4, plectin, BP180 and BP230. In the merged images (C,F,I) plectin, BP180 and BP230 are in green, and ß4 is in red. Co-localized staining appeared in yellow. Note that prevention of the interaction between ß4 and plectin results in a diminished localization of BP180 and BP230 in HD-like structures. Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 9. Model for the assembly of HDs in cultured keratinocytes. The association of the 6ß4 integrin with plectin via the ß4 subunit and their clustering is crucial for the formation of type II HDs. When BP180 is present, it will be incorporated into this complex through interactions with both ß4 and plectin. This is followed by the recruitment of additional plectin and BP180 molecules, resulting in a further increase of the size and the stability of the ternary complexes. In a final step, BP230 becomes incorporated into these complexes, and the HD-like structures containing 6ß4, BP180 and plectin are turned into type I HDs. Because both plectin and BP230 interact with the same site on BP180, it is likely that they may compete for this binding site.