Site-directed Mutagenesis within an Ectoplasmic ATPase Consensus Sequence Abrogates the Cell Aggregating Properties of the Rat Liver Canalicular Bile Acid Transporter/Ecto-ATPase/Cell CAM 105 and Carcinoembryonic Antigen

Abstract

Processing of DNA damage by the DNA double-strand break repair pathway in mammalian cells is accomplished by multiprotein complexes. However, the nature of these complexes and details of the molecular interactions are not fully understood. Interaction of the yeast RAD51 and RAD52 proteins plays a crucial role in yeast DNA homologous recombination and DNA double-strand break repair. Here, specific interactions between human RAD51 and RAD52 proteins are demonstrated both in vivo, using the yeast two-hybrid system and immunoprecipitation of insect cells co-infected with RAD51 and RAD52 recombinant viruses, and in vitro, using affinity chromatography with purified recombinant proteins. These results suggest that RAD52 may modulate the catalytic activities of RAD51 protein such as homologous pairing and strand exchange through a direct physical interaction. In addition, the domain in RAD52 that mediates this interaction was determined in vitro and in vivo. The RAD51-interacting region (amino acids 291-330) of the human RAD52 protein shows no homology with the yeast RAD52 protein, indicating that the interaction between RAD51 and RAD52 is species-specific.

Reconstitution of Bile Acid Transport in a Heterologous Cell by Cotransfection of Transporters for Bile Acid Uptake and Efflux

“…This may mean that it is a member of a nonconventional class of transporters that only have a single transmembrane domain and includes the minK potassium channel (31,32) and an influenza virus M2 proton pump (33,34). However, our recent studies of the cell adhesion properties of CBATP in infected Sf9 cells (39) have indicated that CBATP molecules bind to each other. Clustering of CBATP molecules could, therefore, bring two or more CBATP molecules and their transmembrane domains into close proximity to potentially form a pore in the membrane for bile acid efflux.…”

Two Variable Regions in Carcinoembryonic Antigen-related Cell Adhesion Molecule1 N-terminal Domains Located in or Next to Monoclonal Antibody and Adhesion Epitopes Show Evidence of Recombination in Rat but Not in Human

“…3), two regions of sequence variability (V-regions) were detected. Moreover, these V-regions were located adjacent to or within the regions corresponding to binding sites identified previously for opa proteins (55,56) and MHV (57-59) and adhesive domains mediating intercellular adhesion (25,28,33) (Fig. 3).…”

“…Epitope B Is Located within the Major Adhesive Site in the CEACAM1 a N-domain-In previous reports (25,28,33,(55)(56)(57)(58)(59), the adhesive sites for CEA and the binding domains for MHV and opa proteins were localized to the C ␤-strand and CCЈ loop domains. Because one of the mAb epitopes was within this region, it was of interest to determine whether the two mAbs could block cell adhesion.…”

“…Surprisingly, however, there have only been a few studies of this nature for rat CEACAM1. Sippel et al (25), for example, demonstrated that mutation of Ser-503 abrogated CEACAM1 a -mediated aggregation, whereas Estrera et al (29) determined that phosphorylation of Ser-503 was essential for tumor suppression. To gain further insight into the characteristics and origin of Ceacam1 N-domains, we have utilized genomic PCR to search the rat genome for additional N-domain encoding sequences.…”

“…Relatively recent studies have also shown that the ectodomain is not required for tumor suppression; the CEACAM1 a -4L cyto domain is necessary and sufficient for inhibiting tumorigenicity (24). Cell-cell adhesion, on the other hand, appears to be an activity that is mediated primarily by the N-terminal V-like domain (4,(25)(26)(27). Because all of the CEACAM1 alleles and genes have…”