title: Immunochemical characterization of two isoforms of rat liver ecto-ATPase that show an immunological and structural identity with a glycoprotein cell-adhesion molecule with Mr 105,000.
abstract: One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The association of ATPase activity with cell-CAM105 raises the possibility that extracellular nucleotides may play important roles in regulating cell adhesion.
authors.affiliation: Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
authors.forename: S H
authors.initials: SH
authors.lastname: Lin
authors.forename: O
authors.initials: O
authors.lastname: Culic
authors.forename: D
authors.initials: D
authors.lastname: Flanagan
authors.forename: D C
authors.initials: DC
authors.lastname: Hixson
mesh_terms: D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000737:chemistry; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D051381:Rats; D014162:Transfection
chemical_list: D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D010446:Peptide Fragments; D004247:DNA; D000251:Adenosine Triphosphatases