Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.

Abstract

Abstract. Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed ct-, ~-, and qc-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/ot-catenin, and either/3-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, H-catenin, and plakoglobin cosedimented as complexes. t~-Catenin was not associated with these complexes after synthesis, but a subpopulation of a-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin//3-catenin, and cadherin/ plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of t~-catenin. Subsequently, >90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction. These results indicate that catenins either interact with cadherin complexes synthesized at different times, or there was exchange of catenins between labeled and unlabeled cadherin complexes. In addition, cross-linking revealed additional proteins associated with catenin complexes. We propose a model in which the assembly of cadherin/catenin complexes is regulated by the exchange of catenin...

Hepatocyte Growth Factor Alters the Polarity of Madin-Darby Canine Kidney Cell Monolayers

“…It should be noted that at 24 h, there is only a very small amount of apically accessible Ecadherin, which does not become prominent until 48 -72 h. Therefore, the response to HGF can be viewed as having two phases: 1) an early phase that peaks at 24 h and is characterized primarily by an increase in the amount of E-cadherin associated with ␤-catenin; and 2) a late phase that peaks at 48 -72 h and is characterized by the return of both ␤-catenin synthetic rate and association between ␤-catenin and E-cadherin to baseline levels, and a large amount of apically accessible E-cadherin. Assembly of E-cadherin/␤-catenin complexes in polarized MDCK cells is a dynamic process (20) involving existing and newly synthesized components. Therefore, it is possible that HGF modifies this assembly process either through alteration in cadherin and catenin synthetic rates and/or modification of existing cadherins and catenins.…”

“…Metabolic Labeling-Filter-grown cells were starved in MEM lacking cysteine and methionine (obtained from UCSF Cell Culture Facility and supplemented with 5% fetal bovine serum for 20 min, then pulselabeled for 15 min in the same medium supplemented with [ 35 S]cysteine and [ 35 S]methionine (250 Ci/ml Tran 35 Slabel, ICN Biochemicals, Irvine, CA) as described previously (20). 35 S incorporation into immunoprecipitated proteins was analyzed by SDS-PAGE and quantitated with a PhosphorImager (Molecular Dynamics).…”

Changing Roles of Cadherins and Catenins during Progression of Squamous Intraepithelial Lesions in the Uterine Cervix

“…17 Immunoprecipitated E-cadherin complexes were analyzed for the presence of catenins in immunoblotting ( Figure 6E). In E-cadherin immunoprecipitates a tremendous decrease was detected for ␣-catenin, ␤-catenin, and plakoglobin associated with E-cadherin, increasing with grade of SIL.…”

“…Cross-linking of the molecules of the cadherin-catenin complex was performed after the method of Hinck et al, 17 using dithio-(bis)-succinimidylpropionate (DSP), a cleavable bifunctional linker (Pierce Chemical Co., Rockford, IL). Detergent extraction of cells (1% Triton X100 or 25 18 nonextracted cell fraction in 1% sodium dodecyl sulfate, immunoprecipitation, and immunoblotting were as previously described.…”

“…12 Suggestively originating from the reserve cells, SILs are believed to inherit some differentiation features of their progenitors. SILs express mostly cytokeratins 6,8,17, and 18; in this they resemble reserve cell phenotype/differentiation, although they also express some keratins (5,14,19) common to both the reserve cells and the basal cells of normal squamous epithelium. 14 In this study, we have analyzed immunohistochemically whether the intercellular adhesions typical for the reserve and basal cells are reproduced in SILs cells during the development and progression of these lesions.…”