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.

CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer

“…On the basis of such results, a tumor suppressive role for CEACAM1 has been postulated. Conformingly, CEACAM1 overexpression in different prostate cancer cell lines used in experimental tumor models in mice reduced tumor growth without affecting the proliferation of tumor cells themselves (Lin et al, 1991). Furthermore, it has been reported that the tumor inhibitory function of CEACAM1 depends on the cis-determinants in its cytoplasmic domain (Izzi et al, 1999).…”

Association of an 80 kDa protein with C-CAM1 cytoplasmic domain correlates with C-CAM1-mediated growth inhibition

“…C-CAM1 has sequence homology with carcinoembryonic antigen (Aurivillius et al, 1990;Lin et al, 1991). As predicted from its cDNA sequence, C-CAM1 contains four extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic domain.…”

Expression and androgen regulation of C-CAM cell adhesion molecule isoforms in rat dorsal and ventral prostate

“…Control, sham-operated animals; castrated, castrated animals; +TP, castrated animals treated with testosterone propionate; +TP/F, castrated animals treated with testosterone propionate plus 4-hydroxy¯utamide both were used for the immunohistochemical analyses of rat prostate tissues. These included: Ab669, produced by immunizing rabbits with puri®ed C-CAM protein (Lin et al, 1991), that recognizes both the L-and S-C-CAM isoforms; Mab 5.4, a mouse monoclonal antibody of the IgG1 subtype, that also recognizes both isoforms (Hixson and McEntire, 1989); anti-CL, which is a polyclonal rabbit anti-peptide antibody speci®c for the cytoplasmic domain of L-form C-CAM and was generated by immunizing rabbits with keyhold limpet hemacyanin-conjugated peptides (Lin et al, 1991). We also used a rabbit polyclonal antibody anti-CS, produced against the hexapeptide GGSGSF, which is found only in the cytoplasmic domain of S-form C-CAM (Thompson et al, 1994), and monoclonal antibody K903 or 34bE12 (Keratin-903, isotype IgG, Enzo Diagnostics, Farmingdale, NY, USA), which is speci®c for high-molecularweight cytokeratins 1, 5, 10 and 14, and was used to identify the basal cells of rat prostate.…”

“…Structures of the VP were studied in a longitudinal plane. Antibodies diluted in phosphate-bu ered saline (PBS) at optimal concentrations, as determined from the rat liver section assays (Lin et al, 1991), were incubated with rat prostate tissue sections at room temperature. IF labeling using mouse monoclonal antibodies was performed by using the ABC kit (Vector Laboratories) as follows: The sections were sequentially blocked in 10% normal goat serum, avidin D, and biotin for 15 min each to decrease nonspeci®c staining.…”

“…C-CAMs are cell adhesion molecules of immunoglobulin supergene family and structurally di erent from cadherins and integrins (Lin et al, 1991). L-and Sform C-CAM, the predominant isoforms in the rat, are generated from a single gene by alternative splicing (Cheung et al, 1993a;Najjar et al, 1993).…”