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R Kemler

Publications and source records attributed to R Kemler.

At least 19 recordsLinked to original sources
PubMed1992

Classical cadherins.

Cadherins represent a gene family of Ca(2+)-dependent cell adhesion molecules (CAMs) identified during development and in adult organs. They generally mediate cell-cell adhesion by homotypic interaction, although heterotypic binding between different cadherin molecules is possible. Molecular cloning and sequence comparison has led to the characterization of a highly homologous group of 'classical' cadherins and more distantly related members, together composing a gene superfamily. The classical cadherins are transmembrane glycoproteins which exhibit, in addition to the structural homologies, a very similar overall protein topology. Protein sequence comparison has led to the identification of domains of common functional importance. The cytoplasmic domains of cadherins associate with peripheral cytoplasmic proteins termed catenin alpha, beta and gamma with molecular weights of 102, 88 and 80 kDa respectively. This complex formation seems to regulate the adhesive function of cadherins, most likely by connecting cadherins with actin microfilaments. Possible implications of catenins for cadherin function are discussed.

Animals↗
PubMed1992

Molecular organization of the uvomorulin-catenin complex.

The Ca(2+)-dependent cell adhesion molecule uvomorulin is a member of the cadherin gene family. Its cytoplasmic region complexes with structurally defined proteins termed alpha-, beta-, and gamma-catenins. Here we show that A-CAM (N-cadherin), another member of this gene family, also associates with catenins suggesting that this complex formation may be a general property of the cadherins. For uvomorulin it has been found that this association with catenins is of crucial importance for the adhesive function, but little is known about the molecular organization of the uvomorulin-catenin complex. Using a combination of biochemical analyses we show that a single complex is composed of one molecule of uvomorulin, one or two molecules of beta-catenin, and one molecule of alpha-catenin. Furthermore, beta-catenin seems to interact more directly with uvomorulin. In pulse-chase experiments beta-catenin is already associated with the 135-kD uvomorulin precursor molecule but the assembly of the newly synthesized alpha-catenin into the complex is only detected around the time of endoproteolytic processing.

Animals↗
PubMed1991

The structure of the gene coding for the mouse cell adhesion molecule uvomorulin.

We have recently shown that the Ca2+ dependent cell adhesion molecule uvomorulin is encoded by a single gene, localized on mouse chromosome 8. Here we describe the organization of the uvomorulin gene and give an initial characterization of the uvomorulin promoter. Uvomorulin is encoded by 16 exons, which are distributed over a region of more than 40 kb genomic DNA. The exon structure of the genes for uvomorulin and its chicken homologue L-CAM are nearly identical and thus highly conserved. The relationship between the exon structure and the structure of the uvomorulin protein is analysed. The initiation site of transcription of the uvomorulin gene is located 127 bp upstream of the translation start site in a GC-rich region with no TATA-box, but with a GC-box in position -48 and a CCAAT-box starting at position -65 with respect to the transcription start site. 1.6 kb of the uvomorulin promoter (-1492 to + 92) confer cell type specific promoter activity to the CAT reporter gene. Homologies to known cis acting elements of other promoters are discussed.

3T3 Cells↗
PubMed1991

The uvomorulin-anchorage protein alpha catenin is a vinculin homologue.

The cytoplasmic region of the Ca(2+)-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed alpha, beta, and gamma catenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against alpha catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that alpha catenin is complexed with other cadherins. In immunofluorescence tests, alpha catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk- cells, alpha catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of alpha catenin at membrane areas of cell contacts. We also have cloned and sequenced murine alpha catenin. The deduced amino acid sequence reveals a significant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.

Amino Acid Sequence↗
PubMed1991

Overexpression of uvomorulin in a compaction-negative F9 mutant cell line.

The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.

Animals↗
PubMed1991

Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos.

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.

Animals↗
PubMed1991

Cell adhesion molecule uvomorulin expression in human breast cancer cell lines: relationship to morphology and invasive capacities.

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.

Blotting, Northern↗
PubMed1990

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function.

We show that a synthetic peptide corresponding to the sequence of one putative Ca2+ binding motif of the cell adhesion molecule uvomorulin is able to complex Ca2+. This function is abolished if the first Asp in the peptide is replaced by Lys. Accordingly, we expressed in L cells mutant uvomorulin with a replacement of Asp to Lys or Ala. Mutant protein was resistant to Ca2+/trypsin under mild conditions but became susceptible at or near the site of replacement at higher concentrations, leaving the remaining Ca2+ binding domains protected. Remarkably, in cell aggregation assays both mutant uvomorulins failed to mediate cell adhesiveness, demonstrating that a single amino acid substitution in one Ca2+ binding site inactivates the adhesive function.

Amino Acid Sequence↗
PubMed1990

Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity.

Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.

Animals↗
PubMed1990

Altered uvomorulin expression in a noncompacting mutant cell line of F9 embryonal carcinoma cells.

Uvomorulin (E-cadherin) is a cell adhesive molecule analogous to L-CAM in the chicken. Uvomorulin is important in the process of compaction in eight-cell mouse embryos and plays a role in F9 embryonal carcinoma cell interactions but it is not clear if it mediates aggregation or compaction, the closer interaction that also occurs in F9 cells cultured in suspension. This paper describes the finding of reduced levels of uvomorulin in a mutant cell line of F9 (5.51 att-) that is consistent with a role for uvomorulin in both aggregation and compaction. The mutant line expresses 40-50% of normal levels of uvomorulin as measured by surface radioiodination, immunoblotting, and biosynthetic labeling and immunoprecipitation with two different antisera. The mutant cell line expresses only abnormally unstable uvomorulin transcripts at very low levels. In addition, karyotypic analyses revealed an abnormal chromosome 8 on which the uvomorulin gene is located and therefore could account for aberrant uvomorulin expression. F9 5.51 att- cells aggregate loosely but do not compact (A. Grover, M. J. Rosenstraus, B. Sterman, M. E. Snook, and E. D. Anderson, 1987, Dev. Biol. 119, 1-11). The conclusion is that reduced levels of uvomorulin are sufficient for aggregation but insufficient for compaction.

Animals↗
PubMed1990

A possible new adhesive site in the cell-adhesion molecule uvomorulin.

The calcium-dependent cell-adhesion molecule uvomorulin is a member of the cadherin gene family. Recent studies on the homophilic binding of molecules from neighbouring cells have shown that the amino-terminal part of these proteins plays an important role in the adhesive mechanism. We show here that the epitope for monoclonal antibody DECMA-1, capable of blocking uvomorulin function, is located close to the membrane proximal part of the extracellular domain. To test the effect of structural changes in this membrane proximal region on the adhesive function of uvomorulin, we have studied the cluster of cysteine residues located in the vicinity of the DECMA-1 epitope. Treatment of cells with the reducing agent dithiothreitol (DTT) cleaved the di-sulphide bonds in uvomorulin and affected the adhesive properties of cells. Close cell-cell contacts accompanied by cell flattening and changes in cell shape were blocked by DTT; however, cell aggregation was not inhibited. Consistent with this, uvomorulin became more susceptible in its membrane proximal part to trypsin digestion after treatment with DTT, indicating that conformational changes in this region of the molecule affect the adhesive function. These results suggest that the membrane proximal region of uvomorulin is involved in the adhesive mechanism.

Animals↗
PubMed1990

Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule.

We have recently found that the cytoplasmic region of the cell adhesion molecule uvomorulin associates with three proteins named catenin alpha, beta, and gamma. Here we show by analysis of various mutant uvomorulin polypeptides expressed in mouse L cells that this association is mediated by a specific domain in the cytoplasmic region. A specific recognition site for catenins is located in a 72-amino acid domain. Interestingly, 69 of the 72 amino acid residues are encoded by a single exon of the uvomorulin gene. To demonstrate the direct interaction between catenins and the 72-amino acid domain, cDNA constructs composed of H-2Kd cDNA and various 3' sequences of uvomorulin were expressed in L cells. Chimeric proteins between H-2Kd and the 72-amino acid domain of uvomorulin were shown, by immunoprecipitation with anti-H-2Kd antibodies, to complex with catenin alpha, beta, and gamma. Catenins connect uvomorulin to cytoskeletal structures. We provide biochemical evidence for an association of the uvomorulin-catenin complex with actin bundles. Our results suggest that catenin alpha plays a key role in the association with actin filaments, whereas catenin beta binds more directly to the cytoplasmic region of uvomorulin. In cell aggregation assays with transfected cells expressing normal or mutant uvomorulin, the adhesive function was expressed only when uvomorulin was associated with catenins. From these results we conclude that the cytoplasmic anchorage of uvomorulin is of major biological importance.

Actins↗
PubMed1990

Correct proteolytic cleavage is required for the cell adhesive function of uvomorulin.

All Ca2(+)-dependent cell adhesion molecules are synthesized as precursor polypeptides followed by a series of posttranslational modifications including proteolytic cleavage. The mature proteins are formed intracellularly and transported to the cell surface. For uvomorulin the precursor segment is composed of 129-amino acid residues which are cleaved off to generate the 120-kD mature protein. To elucidate the role of proteolytic processing, we constructed cDNAs encoding mutant uvomorulin that could no longer be processed by endogenous proteolytic enzymes and expressed the mutant polypeptides in L cells. Instead of the recognition sites for endogenous proteases, these mutants contained either a recognition site of serum coagulation factor Xa or a new trypsin cleavage site. The intracellular proteolytic processing of mutant polypeptides was inhibited in both cases. The unprocessed polypeptides were efficiently expressed on the cell surface and had other features in common with mature uvomorulin, such as complex formation with catenins and Ca2(+)-dependent resistance to proteolytic degradation. However, cells expressing unprocessed polypeptides showed no uvomorulin-mediated adhesive function. Treatment of the mutant proteins with the respective proteases results in cleavage of the precursor region and the activation of uvomorulin function. However, other proteases although removing the precursor segment were ineffective in activating the adhesive function. These results indicate that correct processing is required for uvomorulin function and emphasize the importance of the amino-terminal region of mature uvomorulin polypeptide in the molecular mechanism of adhesion.

Amino Acid Sequence↗
PubMed1989

The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.

Uvomorulin belongs to the group of Ca2+-dependent cell adhesion molecules, which are integral membrane proteins with several structural features in common. In particular, the cytoplasmic part of these proteins is highly conserved in different species, suggesting a common biological function. To test this assumption we transfected a uvomorulin full-length cDNA into uvomorulin-negative mouse NIH 3T3 and L cells. Immunoprecipitations with anti-uvomorulin antibodies detected, in addition to uvomorulin, three independent proteins of 102, 88 and 80 kd which are of host origin and which form complexes with uvomorulin. Using cDNA constructs coding for uvomorulin with cytoplasmic or extracellular deletions it is shown that the 102, 88 and 80 kd proteins complex with the cytoplasmic domain of uvomorulin. Peptide pattern analysis revealed that these three proteins are identical in different mouse cells. When uvomorulin cDNA was introduced into cell lines from other species, such as human HeLa and avian fibroblasts, the expressed uvomorulin was also associated with endogenous 102, 88 and 80 kd proteins and, moreover, each of these proteins showed structural similarities to the respective mouse molecule. A panel of antibodies specific for known cytoplasmic proteins of mol. wts similar to those of the three proteins did not react with any of the described components. This suggests that the 102, 88 and 80 kd proteins constitute a new group of proteins for which we propose the nomenclature of catenin alpha, beta and gamma respectively. The characterization of these proteins provides a first molecular basis for a possible cytoplasmic anchorage of uvomorulin to the cytoskeleton.

Animals↗