title: 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: Recent studies of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP), a member of the carcinoembryonic antigen (CEA) supergene family, indicate that it is a multifunctional protein possessing bile acid efflux, ecto-ATPase, and intercellular aggregating properties. Cheung et al. (Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303-24310) have shown that the amino-terminal Ig V-like domain of this protein is required for its aggregating properties, much like the homologous amino-terminal domain of CEA is required for its aggregating properties. The amino-terminal domains of both CBATP and CEA include a consensus ATPase sequence. Site-directed mutagenesis within this ATPase consensus sequence completely eliminates the ecto-ATPase activity of CBATP (Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820-2826). In this study we examined the possibility that it is this ATPase consensus sequence which is required for the cell aggregating properties of CBATP and CEA and whether there is a relationship between ATPase, aggregating, and bile acid efflux activities. For this we used a baculovirus vector to express in Sf9 cells wild type as well as mutant and chimeric CBATP and CEA molecules. The results indicate that Arg-98 in the ATPase consensus sequence of CBATP and the corresponding residue of CEA are essential for the aggregating properties of these molecules. Moreover Arg-98 is essential for CBATP to interact with itself, CEA to interact with itself, and CBATP to interact with CEA. However, the role of Arg-98 in aggregation is distinct from its role in ecto-ATPase activity and the aggregating properties cannot be attributed to a change in ATP metabolism in the pericellular milieu.
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. perlmutter@kids.wustl.edu
authors.forename: C J
authors.initials: CJ
authors.lastname: Sippel
authors.forename: T
authors.initials: T
authors.lastname: Shen
authors.forename: D H
authors.initials: DH
authors.lastname: Perlmutter
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D002272:Carcinoembryonic Antigen / Q000737:chemistry* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism; D002449:Cell Aggregation; D016384:Consensus Sequence; D008099:Liver; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D018411:Spodoptera; D013329:Structure-Activity Relationship
chemical_list: D015703:Antigens, CD; C069847:CD66 antigens; D002272:Carcinoembryonic Antigen; D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases