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Biomedical subjects

K J Green

Publications and source records attributed to K J Green.

At least 37 records · Page 2Linked to original sources
PubMed1998

Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.

The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing beta-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.

Cadherins↗
PubMed1997

The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell-cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell-cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin-plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

Animals↗
PubMed1997

Two-hybrid analysis reveals fundamental differences in direct interactions between desmoplakin and cell type-specific intermediate filaments.

Desmosomes are cell junctions that act as sites of strong intercellular adhesion and also serve to anchor the intermediate filament (IF) cytoskeleton to the plasma membrane of a variety of cell types. Previous studies demonstrated that the COOH terminus of the desmosomal plaque protein, desmoplakin (DP), is required for the association of DP with IF networks in cultured cells and that this domain interacts directly with type II epidermal keratin polypeptides in vitro. However, these studies left open the question of how desmosomes might anchor other IF types known to associate with these junctions. In this report we used yeast two-hybrid and in vitro dot blot assays to further examine the requirements for direct interactions between desmoplakin and various IF types. Our results confirm the ability of the DP COOH terminus (DPCT) to interact with at least two regions of the head domain of the type II epidermal keratin K1 and also demonstrate that DPCT can interact with the type III IF family members, vimentin and desmin, as well as simple epithelial keratins. Unlike the situation for type II epidermal keratins, the interaction between DPCT and simple epithelial keratins appears to depend on heterodimerization of the type I and II keratin polypeptides, since both are required to detect an interaction. Furthermore, although the interaction between DPCT and K1 requires the keratin head domain, deletion of this domain from the simple epithelial keratins does not compromise interaction with DPCT. The interaction between DPCT and type III or simple epithelial keratins also appeared to be less robust than that between DPCT and K1. In the case of K8/K18, however, the interaction as assessed by yeast two-hybrid assays increased 9-fold when a serine located in a protein kinase A consensus phosphorylation site 23 residues from the end of DP was altered to a glycine. Taken together, these data indicate that DP interacts directly with different IF types in specific ways.

Amino Acid Sequence↗
PubMed1997

Roles of plakoglobin end domains in desmosome assembly.

Plakoglobin, a member of the armadillo family of proteins, is a component of intercellular adhesive junctions. The central domain of plakoglobin comprises a highly conserved series of armadillo repeats that facilitate its association with either desmosomal or classic cadherins, or with cytosolic proteins such as the tumor suppressor gene product adenomatous polyposis coli. Sequences in the N- and C-terminal domains of plakoglobin are less highly conserved, and their possible roles in regulating plakoglobin's subcellular distribution and junction assembly are still unclear. Here we have examined the role of plakoglobin end domains by stably expressing constructs lacking the N and/or C terminus of plakoglobin in A-431 cells. Our results demonstrate that myc-tagged plakoglobin lacking either end domain is still able to associate with the desmosomal cadherin desmoglein and incorporate into desmosomes. In cell lines that express an N-terminal truncation of plakoglobin, an increase in the cytosolic pool of en-dogenous and ectopic plakoglobin was observed that may reflect an increase in the stability of the protein. Deletion of the N terminus did not have a dramatic effect on the structure of desmosomes in these cells. On the other hand, striking alterations in desmosome morphology were observed in cells expressing C-terminal truncations of plakoglobin. In these cell lines, ectopic plakoglobin incorporated into desmosomes, and extremely long junctions or groups of tandemly linked desmosomes which remained well attached to keratin intermediate filaments, were observed. Together, these results suggest that plakoglobin end domains play a role in regulating its subcellular distribution, and that the presence of the C terminus limits the size of desmosomes, perhaps through regulating protein-protein interactions required for assembly of the desmosomal plaque.

Amino Acid Sequence↗
PubMed1996

Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2-terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.

Cadherins↗
PubMed1996

Desmosomes and hemidesmosomes: structure and function of molecular components.

Desmosomes and hemidesmosomes are the major cell surface attachment sites for intermediate filaments at cell-cell and cell-substrate contacts, respectively. The transmembrane molecules of the desmosome belong to the cadherin family of calcium-dependent adhesion molecules, whereas those in the hemidesmosome include the integrin class of cell matrix receptors. In each junction, the cytoplasmic domains of certain transmembrane junction components contain unusually long carboxy-terminal tails not found in those family members involved in linkage of actin to the cell surface. These domains are thought to be important for the regulation of junction assembly and specific attachment of intermediate filaments via associated adapter proteins. Recent developments have suggested the exciting possibility that these junctions, in addition to playing an important structural function in tissue integrity, are both acceptors and affectors of cell signaling pathways. Many desmosomal and hemidesmosomal constituents are phosphoproteins and in certain cases the function of specific phosphorylation sites in regulating protein-protein interactions is being uncovered. In addition, a more active role in transmitting signals that control morphogenesis during development and possibly even regulate cell growth and differentiation are being defined for cytoplasmic and membrane components of these junctions.

Animals↗
PubMed1996

Analysis of desmosomal cadherin-adhesive function and stoichiometry of desmosomal cadherin-plakoglobin complexes.

Desmosomes are intercellular adhesive junctions that associate with the intermediate filament cytoskeleton. The two major classes of transmembrane desmosomal glycoproteins, desmogleins and desmocollins, are widely considered to function as adhesion molecules. This assumption is based in part on their homology to the cadherin family of calcium-dependent homophilic adhesion molecules. In addition, autoantibodies from pemphigus patients bind directly to desmoglein family members and are thought to cause epidermal blistering by inhibiting the function of these cadherins. To directly test the ability of the desmosomal cadherins to mediate adhesion, desmoglein-1 (Dsg1), desmocollin-2 (Dsc2a) and plakoglobin were expressed in mouse L cell fibroblasts. Similar to catenin:classical cadherin complexes, plakoglobin:Dsc2a complexes exhibited an approximately 1:1 stoichiometry; however, plakoglobin:Dsg1 complexes exhibited a 6:1 stoichiometry. When L cells expressing the desmosomal cadherins were tested for the ability to aggregate in suspension, L cells expressing E-cadherin exhibited extensive aggregation, but L cells expressing Dsg1 or Dsc2a did not aggregate. In addition, L cells co-expressing Dsg1, Dsc2a, and plakoglobin failed to aggregate. The cytoplasmic domain of E-cadherin is thought to play a central role in the adhesive function of E-cadherin by providing a link to the actin cytoskeleton. Therefore, two chimeric cadherins comprising the cytoplasmic domain of E-cadherin and the extracellular domain of either Dsg1 or Dsc2a were expressed in L cells. Both chimeras formed a complex with alpha- and beta-catenin. Nevertheless, neither of these chimeras supported aggregation of L cells when expressed individually or when co-expressed. These data suggest that the extracellular domains of the desmosomal cadherins exhibit functional properties distinct from those of the classical cadherins, such as E-cadherin.

Animals↗
PubMed1995

Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1.

To identify regions of the Desmoglein-1 (Dsg1) extracellular domain that are targeted by pemphigus antisera, cDNA sequences encoding various regions of the Dsg1 extracellular domain were ligated to sequences encoding the E-cadherin extracellular anchor and transmembrane and cytoplasmic domains. These constructs were then expressed in mammalian cells, and pemphigus sera were tested for the ability to recognize the Dsg1 extracellular domains. When analyzed by immunoblot, very few pemphigus foliaceus sera recognized the Dsg1 domains. To determine whether pemphigus sera recognize non-denatured Dsg1 domains, constructs were expressed in cultured cells and tested for reactivity with pemphigus sera using live-cell immunofluorescence. The pemphigus foliaceus sera reacted strongly with the Dsg1 extracellular domain by live-cell immunofluorescence and recognized predominantly the amino-terminal region of the Dsg1 extracellular domain. In addition, sera from patients with pemphigus vulgaris also demonstrated strong reactivity with the Dsg1 extracellular domain when tested using live-cell immunofluorescence. In contrast, sera from normal human controls and sera from bullous pemphigoid patients did not react with the Dsg1 extracellular domain. These data indicate that both pemphigus foliaceus and pemphigus vulgaris sera react with conformationally sensitive epitopes in the amino-terminal region of Dsg1.

Base Sequence↗
PubMed1995

Antigen-specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus.

Patients with the autoimmune blistering disease pemphigus foliaceus (PF) have circulating autoantibodies directed against the desmosomal cadherin desmoglein 1 (Dsg1). Based on the fact that purified IgG fractions from PF patients induce loss of cell adhesion in organ culture and in a neonatal mouse model, it has been proposed that these anti-Dsg1 antibodies play a pathogenic role in blister formation. To directly address whether antibodies in PF sera specific for the Dsg1 extracellular domain are indeed pathogenic in the disease, PFIg, a chimeric protein containing the entire extracellular domain of human Dsg1 and the constant region of human IgG1, was produced by baculovirus expression. Incubation of PF patients' sera with the PFIg baculoprotein removed the immunoreactivity of autoantibodies against keratinocyte cell surfaces in all 20 PF and eight Brazilian PF patients' sera tested. This adsorption was conformation dependent, because PFIg protein denatured by low pH or heat was no longer able to adsorb the immunoreactivity of PF sera. Furthermore, the incubation with the PFIg baculoprotein eliminated the pathogenic activity of PF patients' sera and prevented gross blister formation in a neonatal mouse model of pemphigus. Anti-Dsg1 antibodies eluted from the PFIg protein column were pathogenic as they resulted in the appearance of gross blisters in neonatal mice with typical histologic findings of PF. These observations indicate that the extracellular domain of Dsg1 expressed by baculovirus is capable of specifically immunoadsorbing pathogenic autoantibodies from PF patients' sera and provide direct evidence that the anti-Dsg1 autoantibodies in PF sera are indeed pathogenic. The availability of this Dsg1 recombinant protein may facilitate the development of antigen-specific plasmapheresis as a novel therapeutic strategy in pemphigus.

Animals↗
PubMed1994

Posttranslational regulation of plakoglobin expression. Influence of the desmosomal cadherins on plakoglobin metabolic stability.

Desmosomes are adhesive intercellular junctions that act as cell surface attachment sites for intermediate filaments. The desmosomal glycoproteins, desmogleins and desmocollins, are members of the cadherin family of adhesion molecules. In addition, desmoglein has been shown to coimmunoprecipitate with the junctional protein plakoglobin. To characterize further the interaction between plakoglobin and the desmosomal cadherins, stable mouse fibroblast (L-cells) cell lines were generated that express plakoglobin, desmoglein and plakoglobin, or desmocollin and plakoglobin. L-cell lines transfected with a plasmid encoding human plakoglobin expressed plakoglobin mRNA but very little plakoglobin protein. However, plakoglobin protein was expressed at high levels in L-cells coexpressing either desmoglein or desmocollin. In addition, both desmocollin and desmoglein were found to coimmunoprecipitate with plakoglobin. The transient expression of desmoglein in L-cell lines expressing plakoglobin mRNA resulted in the formation of a complex between plakoglobin and desmoglein and in the accumulation of plakoglobin protein. Furthermore, the rate of plakoglobin protein degradation was decreased by 15-20-fold in cell lines expressing either desmoglein or desmocollin. These results demonstrate that the desmosomal cadherins posttranslationally regulate plakoglobin expression by decreasing the rate of plakoglobin degradation.

Animals↗
PubMed1994

Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks.

Desmoplakins (DPs) are the most abundant proteins in the innermost portion of the desmosomal plaque and have been proposed to play a role in the attachment of intermediate filaments (IF) to cell-cell contact sites. Our previous results suggest that the globular end domains of DP perform dual functions: first, to target DP to the desmosome via the NH2 terminus and second, to attach IF to the desmosomal plaque via the COOH terminus. When ectopically expressed in most cultured cells, the COOH terminus plus the rod domain (DP. delta N.SerC23) exhibits striking coalignment with keratin IF networks. However, in certain cell types (e.g. PtK2) or in cells treated with forskolin to activate protein kinase A, DP. delta N.SerC23 exhibits a diffuse cytoplasmic distribution. A variant molecule (DP. delta N.GlyC23) in which a serine located 23 amino acids from the COOH terminus is altered to a glycine, thereby disrupting a protein kinase A consensus phosphorylation site, co-localizes with keratin IF networks regardless of cell type or forskolin treatment. Analysis of the phosphopeptide maps of these DP variants and endogenous DP is consistent with the phosphorylation of the serine 23 residues from the COOH terminus. These results suggest that phosphorylation of a specific residue in the DP COOH terminus may negatively regulate its interaction with keratin IF networks.

Amino Acid Sequence↗
PubMed1994

The human genes for desmogleins (DSG1 and DSG3) are located in a small region on chromosome 18q12.

Desmoglein is a transmembrane glycoprotein component of desmosomes in vertebrate epithelial cells. Two of the three currently known desmogleins are the autoantigens of autoimmune skin blistering diseases, pemphigus vulgaris and pemphigus foliaceus, in which autoantibodies cause the loss of cell adhesion of keratinocytes with resultant blister formation. In this study, the genes for two autoantigens (DSG1 for pemphigus foliaceus and DSG3 for pemphigus vulgaris) were mapped on band q12 of human chromosome 18 by fluorescence in situ hybridization. Furthermore, both genes were localized on a 320-kb genomic fragment separated by pulsed-field gel electrophoresis. These results suggest the possibility of a cluster for the desmoglein gene family on chromosome 18.

Animals↗
PubMed1994

Structure and function of desmosomal transmembrane core and plaque molecules.

Desmosomes are intercellular junctions that function in cell-cell adhesion and attachment of intermediate filaments (IF) to the cell surface. Desmogleins and desmocollins are the major components of the transmembrane adhesion complex, whereas desmoplakins (DPs) are the most prominent components of the cytoplasmic plaque. Based on sequence similarity, desmogleins and desmocollins are related to the calcium-dependent homophilic adhesion molecules known as cadherins. Like the classical cadherins, the desmosomal cadherins contain four homologous extracellular domains bearing putative calcium-binding sites, a single transmembrane spanning domain, and a C-terminal cytoplasmic tail. Molecules in the desmoglein subclass contain a unique C-terminal extension within which is found a repeating motif that is predicted to form two beta-strands and two turns. Stable cell lines expressing desmoglein 1 have been generated from normally non-adherent L cell fibroblasts, to study the contribution of this cadherin to desmosomal adhesion. The predicted sequence of desmoplakin (DP) I suggests it will form homodimers comprising a central alpha-helical coiled-coil rod and two globular end domains. The C-terminus contains three regions with significant homology, each of which is made up of a 38-residue motif also found in two other molecules involved in organization of IF, bullous pemphigoid antigen and plectin. Ectopically expressed polypeptides including the C-terminus of DP I specifically align with keratin and vimentin IF in cultured cells, whereas those lacking this domain do not align with IF. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-terminal end are critical for this interaction. These results suggest that the C-terminus of DP plays a role in the attachment of IF to the desmosome and that a specific site is necessary for interaction with keratin IF. A sequence at the most N-terminal end of DP appears to be required for efficient incorporation into the desmosomal plaque. Interestingly, this region has not been reported to be present in the homologous bullous pemphigoid antigen or plectin molecules and may represent a desmosomal targeting sequence.

Animals↗