CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with α3β1 and α6β4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with α3β1 and α6β4. In β4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and α3β1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and α3β1. Transient transfection studies of PA-JEB cells with β4 revealed that the integrin α6β4 becomes incorporated into the α3β1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of α3β1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with α6β4 in hemidesmosomes, whereas its codistribution with α3β1 in focal adhesions becomes partial. The localization of α6β4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using β4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of β4, we found that for recruitment of CD151 into hemidesmosomes, the β4 subunit must be associated with α6, confirming that integrins associate with tetraspans via their α subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin α6β4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

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The integrin ␣6␤4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of ␣6␤4 in live cells two different ␤4-chimeras were stably expressed in ␤4-deficient PA-JEB keratinocytes. One chimera consisted of full-length ␤4 fused to EGFP at its carboxy terminus (␤4-EGFP). In a second chimera the extracellular part of ␤4 was replaced by EGFP (EGFP-␤4), thereby rendering it incapable of associating with ␣6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the ␤4-EGFP and EGFP-␤4 hemidesmosomes disappear, and a proportion of the ␤4-EGFP, but not of the EGFP-␤4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving "footprints" of the migrating cell. PA-JEB cells expressing ␤4-EGFP migrate considerably more slowly than those that express EGFP-␤4. Studies with a ␤4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (␤4 R1281W -EGFP) suggest that the stabilization of the interaction between ␣6␤4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of ␤4 with plectin renders the bond between ␣6␤4 and laminin-5 more stable, i.e., ␤4-EGFP is less dynamic than ␤4 R1281W-EGFP. On the other hand, when ␣6␤4 is bound to laminin-5, the binding dynamics of ␤4 to plectin are increased, i.e., ␤4-EGFP is more dynamic than EGFP-␤4. We suggest that the stability of the interaction between ␣6␤4 and laminin-5 is influenced by the clustering of ␣6␤4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether ␣6␤4 will inhibit cell migration or not. INTRODUCTIONKeratinocytes adhere to the basement membrane by hemidesmosomes that serve as anchoring sites for the intermediate filament system and play a critical role in stabilizing the association of the dermis with the epidermis. The transmembrane components of hemidesmosomes comprise the laminin-5 (LN-5) binding integrin ␣6␤4 and the bullous pemphigoid antigen (BP)180. These proteins are connected via the hemidesmosomal proteins plectin and BP230 to the keratin intermediate filament system (reviewed by Borradori and Sonnenberg, 1999).Based on their structural constituents, two subtypes of hemidesmosomes are distinguished. Type I hemidesmosomes contain ␣6␤4, plectin, BP180, and BP230 (Green and Jones, 1996), whereas type II hemidesmosomes contain only ␣6␤4 and plectin (Uematsu et al., 1994). Recently, the tetraspanin CD151 was identified as another component of both type I and II hemidesmosomes (Sterk et al., 2000). Type I or classical hemidesmosomes are present in basal keratinocytes of squamous and complex epithelia . Type II hemidesmosomes are found in intestinal epi...

The chromosome of Bordetella pertussis harbours a region of 27 contiguous kb, which contains the bvg, fha and fim genes, involved in the co-ordinate regulation of virulence genes, FHA production and fimbriae production, respectively. The linkage of FHA and fimbrial genes has resulted in some confusion concerning the existence and location of genes required for the production of FHA and the function of the fimbrial genes fimB-D, which were proposed to be involved in both FHA and fimbriae biosynthesis. Through the use of non-polar mutations in each of these genes, we found that fimB-D are required for the production of both serotype 2 and 3 fimbriae, but not for FHA biosynthesis. Furthermore, a large open reading frame, designated fhaC, was identified downstream of fimD. It was shown that fhaC is essential for FHA production but not for fimbriae biogenesis. We propose that insertion mutations in fimB-D affect FHA production because of polar effects on fhaC expression. An insertion in the region downstream of fhaC had only a slight effect on FHA and fimbriae production. The fhaC gene product shows homology with ShIB and HpmB, two outer membrane proteins involved in export and activation of the haemolysins, ShIA and HpmA, of Serratia marcescens and Proteus mirabilis, respectively. Homology is also observed between the N-termini of FHA, ShIA and HpmA. Export of the haemolysins requires the N-termini of these molecules, and when this region was removed from FHA by an in-frame deletion, FHA biosynthesis was abolished. These results suggest that the N-terminus of FHA interacts with FhaC, and that as a result FHA is transported across the outer membrane.

HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The therapeutic benefit of HER2-targeting agents, which depend on PI3K/Akt inhibition, can be overcome by hyperactivation of the heregulin (HRG)/HER3 pathway. Here we describe an unbiased phenotypic combinatorial screening approach to identify a bispecific immunoglobulin G1 (IgG1) antibody against HER2 and HER3. In tumor models resistant to HER2-targeting agents, the bispecific IgG1 potently inhibits the HRG/HER3 pathway and downstream PI3K/Akt signaling via a "dock & block" mechanism. This bispecific IgG1 is a potentially effective therapy for breast cancer and other tumors with hyperactivated HRG/HER3 signaling.