Formation of hemidesmosome-like structures in the absence of ligand binding by the (alpha)6(beta)4 integrin requires binding of HD1/plectin to the cytoplasmic domain of the (beta)4 integrin subunit.

Hemidesmosomes are adhesion structures that mediate anchorage of epithelial cells to the underlying basement membrane. We have previously shown that the (alpha)6(beta)4 integrin can induce the assembly of these multi-protein structures independent of binding to its ligand laminin-5 (ligand-independent formation of hemidesmosomes). Our results suggested a role for HD1/plectin, which binds to the cytoplasmic domain of the (beta)4 integrin subunit, in controlling the clustering of hemidesmosomal components at the basal side of the cell. Using keratinocytes derived from patients lacking HD1/plectin, we now show that ligand-independent formation of hemidesmosomal clusters indeed requires HD1/plectin, in contrast to the ligand-dependent assembly of hemidesmosomes. No clustering of the (alpha)6(beta)4 integrin, or of the bullous pemphigoid antigens BP180 and BP230, was seen when HD1/plectin-deficient keratinocytes were plated on fibronectin or type IV collagen. In (&bgr;)4-deficient keratinocytes, expression of an interleukin 2 receptor (IL2R) transmembrane chimera containing the (beta)4 cytoplasmic tail with the mutation R1281W, which abrogates HD1/plectin binding, resulted in a diffuse distribution of the chimeric receptor. In contrast, a (beta)4(R1281W) mutant that can associate with (alpha)6 and bind ligand, was found to be directed to the basal surface of the cells, at sites where laminin-5 was deposited. In addition, this mutant induced clustering of BP180 and BP230 at these sites. Together, these results show that the formation of hemidesmosomes requires binding of either ligand or HD1/plectin to the (beta)4 integrin subunit. Intriguingly, we found that IL2R/(beta)4 chimeras become localized in pre-existing hemidesmosomes of HD1/plectin-deficient keratinocytes, and that this localization requires a domain in the (beta)4 cytoplasmic tail that is also required for HD1/plectin binding (residues 1115-1356). Because this part of (beta)4 lacks the BP180 binding site, and since we show in this study that it is unable to interact with the same part on another (beta)4 molecule, we suggest that the chimera becomes incorporated into hemidesmosomes of HD1/plectin-deficient keratinocytes by interacting with an as yet unidentified hemidesmosomal component.

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

Biology and function of hemidesmosomes.

Hemidesmosomes are cell-substratum adhesion sites that connect the extracellular matrix to the keratin cytoskeleton. Our knowledge of the function of these structures has greatly increased as a result of studies on patients with aberrant expression of hemidesmosome components and studies using targeted inactivation of mouse genes encoding these components. Insight into the formation of hemidesmosomes, as well as into protein-protein interactions that occur in these junctional complexes, has recently been gained by in vitro cell transfections, blot overlay and yeast two-hybrid assays. In addition, recent results indicate that the alpha6 beta4 integrin is involved in the transduction of signals that are induced by the extracellular matrix and which modulate processes as diverse as cell proliferation, differentiation, apoptosis, migration and tissue morphogenesis. Thus it seems that hemidesmosomes do not merely maintain dermo-epidermal adhesion and tissue integrity, but that they are also implicated in intracellular signaling. Here we discuss recently published data on the biology and function of hemidesmosomes.

Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180.

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the beta4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in beta4 expression. We found that the expression of wild-type beta4 restored the ability of the beta4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the beta4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the beta4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between beta4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for alpha6 and beta4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of beta4 with BP180. Nevertheless, beta4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of beta4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of beta4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of beta4 with other hemidesmosomal components, e.g., BP180, is regulated.

Ligand-independent role of the beta 4 integrin subunit in the formation of hemidesmosomes.

Recently, we have shown that a region within the beta4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of alpha6 beta4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the beta4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the beta4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of beta4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/beta4 chimeric constructs in COS-7 and CHO cells, which do not express alpha6 beta4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/beta4 chimeras in a keratinocyte cell line derived from a patient lacking beta4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of alpha6 beta4-expressing cells to fibronectin. These findings establish that the beta4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of alpha6 beta4 to ligand, and heterodimerization of alpha6 with beta4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.

v-Crk-induced cell transformation: changes in focal adhesion composition and signaling.

v-Crk is an oncogene product in which a viral Gag sequence is fused to a cellular Crk sequence. It contains one SH2 and one SH3 domain. To gain insight into the molecular mechanisms underlying v-Crk-induced cell transformation, we studied the subcellular localization and molecular interactions of v-Crk in v-Crk-transformed NIH-3T3 cells. Our results show that v-Crk specifically localizes to focal adhesions where it induces protein tyrosine phosphorylation. Subcellular fractionation studies indicated that a significant amount of v-Crk is present in the cytoskeletal cell fraction, a fraction that includes focal adhesions. Tyrosine phosphorylated proteins, including p130CAS, were also predominantly found in the cytoskeletal fraction. We show that v-Crk induces a translocation of p130CAS to the cytoskeleton, which is accompanied by hyperphosphorylation of this protein. Mutational analyses showed that functional v-Crk SH2 domain is required for the localization of v-Crk in focal adhesions. Functional v-Crk SH2 and SH3 domains were both found to be required for the observed increase in tyrosine phosphorylation of focal adhesion proteins and for the translocation and hyperphosphorylation of p130CAS. v-Crk immunoprecipitation studies revealed that cytoskeleton-associated v-Crk interacts with both p130CAS and an unidentified tyrosine kinase. These findings suggest that formation of a focal adhesion-located complex consisting of v-Crk, a tyrosine kinase and p130CAS, which may lead to the hyperphosphorylation of p130CAS. These specific and localized signaling events may represent initial steps in the process of v-Crk-induced cell transformation.

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains.

Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors.