Summary Cell recognition and adhesion, being of prime importance for the formation and integrity of tissues, are mediated by cell adhesion molecules, which can be divided into several distinct protein superfamilies. The cell adhesion molecule C-CAM (cell-CAM 105) belongs to the immunoglobulin superfamily, and more specifically is a member of the carcinoembryonic antigen (CEA) gene family. C-CAM can mediate adhesion between hepatocytes in vitro in a homophilic, calciumindependent binding reaction. The molecule, which occurs in various isoforms, is expressed in liver, several epithelia, vessel endothelia, platelets and granulocytes and its expression is dynamically regulated under various physiological and pathological conditions. It is proposed that C-CAM in different cells and tissues plays different functional roles, where the common denominator is membrane-membrane binding. Introduction One of the major mysteries of metazoan life is how one single cell can give rise to organisms composed of billions of cells that are organized in a highly specific and very complex manner. This process, known as embryonic development, depends on a number of genetic and epigenetic events that regulate the properties, functions and pattern formation of the developing cells. One factor known to be of importance is contact formation and adhesion between cells and between cells and the extracellular matrix. It is easy to envisage that cell-cell and cell-matrix adhesion take part in the building of tissues from the component cells. It is also reasonable to assume that proper adhesive interactions arc vital for the maintenance of the structure and function of tissues and organs in mature organisms. In order to obtain a better understanding of cell adhesion, researchers have for many years tried to identify and characterize the cell surface molecules and structures that might participate in or mediate cell-tocell and cell-to-matrix adhesion. This has led. during the last decade, to the identification of a number of cell surface proteins that have properties expected for cell adhesion molecules (CAMs) and extracellular matrix receptors (ECM-Rs) , respectively. The strategy in this
search has been to look for molecules that participate in binding of cells to cells or of cells to various ECM components in in vitrn assays. Undoubtedly, the molecules that have becn identified as CAMs and ECM-Rs by these assays are able to bind cells to cells or to ECM components. However. it must be stressed that beyond that property we know very little about the biological functions of these molecules. While some CAMs may have as their prime role creation of physically stable bonds between cells in tissues, others may primarily be involved in other processes such as signalling between cells. Since signalling between cells must involve molecular contacts, it follows that if the binding energy in such contacts is high enough to withstand the disruptive forces in the adhesion assay used, the molecules involved in such interactions will be recognized in the screening for adhesion molecules('). Thus, the definition of CAMs, as we know them today, is an operational one, haTed on the adhesion assay. Other approaches are needed to elucidate the functional roles of these molecules in the structure and functions of tissues.
Cell Adhesion Molecules Several CAMs have been identified. cloned and sequenced and it is apparent that different families of CAMS exist. Thus, the majority of the known CAMs belongs to one of the following families: the immunoglobulin (Tg) superfamily('), the cadhcrin family('), the integrin superfamily(4),the LEC-CAM family(5)and the H-CAM family(5). Before describing these families. a few remarks concerning the binding mechanisms of CAMs should be madc. It has been found that CAMs bind either in a homophilic or a heterophilic manner. In homophilic binding, the CAM species binds to itself, that is one CAM molecule on one cell binds to another identical CAM molecule on an adjacent cell. In heterophilic binding, the CAM molecule binds to another molecule of different identity. Another u5eful property for the characterization of the binding mechanisms is the dependence of calcium ions. Some CAMs, like the cadherins, have an absolute requirement for calcium, whereas others are independent of this divalent cation.
The Ig-superfamily This is a large family that, in addition to the immunoglobulins, contains many other proteins, the majority being involved in cellular recognition phenomena@). Thc common building block is the immunoglobulin domain of about 100 amino acid residues that is arranged as a sandwich of two sheets of anti-parallel pstrands. Different members of the superfamily have varying numbers of Ig-like domains. The prototype for a CAM is N-CAM which has five Ig-like domains in the extracellular portion of the molecule('). N-CAM appears in several isoforms as a result of alternative splicing. Some of these molecules are single-pass
Table 1 . CAMS in the Ig-supevfanrily
Dcmonstrated cell ddhcslond
'') The LEC-CAM family Proteins in the LEC-CAM family (also known as selectins) are found in leukocytes, platelets and vessel endothelial cells(i3”). Mel-14 in lymphocyte5 i \ a homing receptor. GMP-140 occurs in endothelial cells and platelets and probably participates in plateletplatelet binding in late phases of platelet aggregation. ELAM-1 is an endothelial cell protein which can be induccd by inflammation and cytokines These proteins are single-pass transmembrane proteins, which in their extracellular portions have a lectin-like domain (I,). an epidermal growth factor-like domain (E) and a varying number of coniplement binding protein-like domains (C). Hence, they are called LEC-CAM. The H-CAM family Members of the H-CAM family were also originally identified as lymphocyte homing receptors(’). Sequence analysis then demonstrated that related proteins are found in several cell types, including various epithelia and a subset of glia cells. These proteins are single-pass transmembrane proteins which have motifs homologous to proteoglycan core protein and link protein in their extracellular portion. Some of the members of the H-CAM family are proteoglycans themselves and carry chondroitin sulfate chains. One of the HI-CAMS.CD44, is the major receptor for liyalur~nate(’~). C-CAM C-CAM was identified as a i-esult of our systematic search for cell surface molecules that were involved in cell-cell adhesion of isolated rat hepatocytes. The protein that we found was named cell-CAM 105 indicating that it is a cell-cell adhesion molecule with an apparent molecular weight of 105000(”). Later we demonstrated that this molecule also occurs in several other tissues but that its size is not always 105 kD(19). Accordingly, we now refer to this molecule simply as C-CAM. Identification and adhesive properties of C-CAM The original identification of C-CAM utilized an immunological approach(’*). which has also been used in other laboratories to idenlify N-CAM, the cadherins and several other adhesion molecules(’’. After having identified C-CAM we could demonstrate that not only antibodies against C-CAM but also pure C-CAM itself was able to inhibit hepatocyte cell-cell adhesion(’”), The mechanism of action was analyzed by binding experiments performed with pure C-CAM. In blotting experiments with electrophoretically separated plasma membrane proteins from rat liver. it %as found that pure C-CAM bound essentially to only one component. the latter having an apparent molecular weight of
105 000. We thus suspected that C-CAM binds to itself in a homophilic manner. Pure C-CAM was then used both in solution and in reconstituted liposomes to study the binding both to pure C-CAM and to isolated hepatocytes, and we demonstrated that C-CAM, indeed, can bind to itself in a homophilic, calciumindependent rnanned’l). So far we have not found any other ligand for C-CAM that is exposed on the extracellular surface of hepatocyte plasma membranes. C-CAM accordingly has the potential of mediating cell-cell or membrane-membrane binding in vivo. Chemical structure of liver C-CAM C-CAM purified from rat liver appears as two chains with apparent molecular weights of 105000 and 110 000, respectively(20). The smaller chain always occurs in larger amounts than the larger one. Peptide mapping has demonstrated that the two chains are structurally similar(’*). Both chains are highly glycosylated, carrying only N-linked oligosaccharides, most of which is of a com lex type in which bi-antennary structures dominate?’*). Oligomannose-t pe structures also comprise a substantial proportion(d C-CAM is an amphipathic molecule which can be solubilized in intact form only by detergents. Partition experiments have shown that the intact molecule exclusively ends up in the detergent phase of Triton X-114(”). The biochemical properties are thus in accordance with C-CAM being a transmembrane cell surface protein in hepatocytes. The complete amino acid sequence of rat liver C-CAM has recently been establi~hed(*~). Peptides of purified C-CAM, generated by trypsin cleavement and purification by reversed hase HPLC, were sequenced by Edman degradation(*i? ). The sequences of four such peptides were found to be identical with the amino acid sequence of a recently cloned and sequenced rat liver membrane protein that has been described as an ectoATPase(14).A detailed biochemical analysis confirmed the identity between C-CAM and the ecto-ATPase(”). We have not yet been able to determine which of the two chains of C-CAM that corresponds to the published sequence of the ecto-ATPase. The sequence data confirms that C-CAM is a transmembrane protein with a large N-terminal extracellular portion, a 25 amino acid long transmembrane domain and a 71 amino acid long C-terminal cytoplasmic domain. The protein contains a leading hydrophobic, putative signal sequence. However, since the N-terminal residue was blocked we could not determine the complete N-terminal sequence of the mature protein. A likely possibility is that the signal sequence is cleaved off, leaving a glutamine in the N-terminal position, which would also explain the N-terminal blocking. In that case, the extracellular domain would consist of 389 amino acids and the total, mature protein would contain 485 amino acids. Sixteen potential sites for N-glycosylation are found in the extracellular domain, a finding that agrees well with the chemical
Fig. 1. Linear structure of C-CAM. From the deduced amino acid sequence a linear structure composed of several distinct domains can he predicted. The protein contains two hydrophobic domains, one putative signal sequcnce in thc N-terminal end (indicated by a dashed line) and one putative transmembrane domain (M) near the C-terminal end. Four immunoglobulin-likc domains can bc prcdicted; the most N-terminal one is a V-like domain, the other three are C-2 domains. The C-2 domains, but not the V-like domain, have cysteine residues that could be involved in intradomain disulfide linkages. A consensus sequence for CAMP-dependent phosphorylation (8) is found close to the C-terminal end. The protein contains 36 sites for N-linked glycosylation ( 7 ). The location of the glycosylation sites suggests that the major, N-terminal portion of C-CAM is exposed on the extracellular side, and that the C-terminal end is located on the cytoplasmic side of the plasma membrane.
determination of the carbohydrate content in the mature protein. C-CAM is a member of the immunoglobulin superfamily and the CEA gene family A closer examination of the amino acid sequence of C-CAM revealed that it is a member of the immunoglobulin superfamily (Fig. 1). The sequence suggests that it has four Ig-domains in the extracellular portion(’3). The most N-terminal one is a V-like domain and the other three, which have conserved cysteins that might form intradomain disulfide bonds have consensus sequences indicating that they are C2-like domains. Furthermore. Lin and Guidotli noted that the ectoATPase/C-CAM is highly homologous to human biliary glycoprotein 1 $BGPl 1 , with . 65% - of the amino acids being identical( ‘j. BGPl is a member of the CEAThus, we can conclude that C-CAM belongs to the CEA-subfamily of the immunoglobulin superfamily. Cellular location and tissue prevalence of C-CAM C-CAM was originally identified in hepatocytes(’*). With the availability of specific antibodies, it became possible to map its tissue distribution and subcellular location. We then found that C-CAM antigens are expressed in a number of organs of mature rats(”). The tissue distribution within these organs is, however, limited. Thus, C-CAM is primarily expressed in various epithelia (including hepatocytes of the liver), endothelia of capillaries, small arteries and veins, and in megakaryocytes, platelets, polymorphonuclear leukocytes and a subset of mononuclear leukocytes(’’). It has not been found in nervous tissues, muscle tissues or connective tissues. Radioimmunochemical analyses indicated immunolo ical identity between C-CAM in the various organs( ’). However, immunoblotting of
Fig. 2. Subcellular localization of C-CAM. Iininunohistochcmistry has demonstrated that C-CAM has different locations in different cell types. Thc occurrence of C-CAM in some representative cell types is indicated by hcavy lines at locations indicated by arrows. (A) In mature liver C-CAM is highly conccntrated in the membranes of the bile canaliculi. (B) In cultures of epithelial cells such as NBT-I1 cells and MDCK cells C-CAM is found in the lateral membranes where thc cells are in contact with each other. (C) In stratified epithelia C-CAM is locatcd in thc ccll-cell borders of suprabasal cells. (D) In brush bordcr-containing cells, such as the epithelial cells of the small intestine and of the renal proximal tubules, C-CAM is localized to the membranes of the microvilli of the brush border. (E) In unactivated platclets C-CAM is found in intracellular sites. Upon activation and aggregation C-CAM becomcs readily accessible on the surface of the aggregates, indicating that it has been redistributed to thc surfacc membrane of the platelets.
electrophoretically separated tissue extracts demonstrated some size variations in C-CAM from the different sources(19).It is presently not known if this reflects differences in the protein chain or in the glycosylation, or if it is a combination of both. By immunohistochemical staining techniques, we found interesting differences in the subcellular location of C-CAM in different cell types (Fig. 2). Essentially the following three different locations have been observed: 1) lateral cell surface membranes; 2) microvillar membranes in apical cell surfaces; 3) intracellular cytoplasmic sites. In mature liver. C-CAM is highly concentrated to the microvillar membranes of bile canaliculi(")). In isolated hepatocytes, C-CAM spreads over the whole cell surface but during culture, it first becomes concentrated to areas of cell-cell contact and later is also found in reformed bile canaliculi(2h).A similar dynamic pattern is observed in hepatocytes of perinatal and regenerating livers, where C-CAM is located at lateral borders of cell-cell contact and subse uently is found primarily in mature bile canaliculi(-7751. In brush border-carrying simple epithelial cells, such as those of small intestinal mucosa and proximal renal tubules, C-CAM is found in high concentrations in the microvillar membranes of the brush borders("). In this location, C-CAM may be important for proper organization of the microvilli of the brush border (Fig. 3). In other epithelia, such as the stratified epithelia of the tongue or the vaginal mucosa. C-CAM is located at the lateral borders of the su rabasal epithelial cells where they contact each othert9). In MDCK cells (dog epithelial cells of renal origin) and NBT-I1 cells (a rat bladder carcinoma cell line) cultured in vitro, C-CAM
Fig. 3. Tentative role of C-CAM in brush border organization. The homophilic binding properties of C-CAM and thc localization to the brush borders suggest that C-CAM can participate in brush border organization by cross-linking of adjacciit inicrovillar membranes. (A) A longitudinal section demonstrating membrane-membrane binding between apical microvilli in addition to membrane-membrane binding of adjacent cells at the basolateral surfaccs. (B) A horizontal section through the microvilli of the brush border. The microvilli are organized in a hexagonal pattern. This might be a result of crosslinking between neighbouring microvilli mediated by homophilic C-CAM binding. The picture demonstrates that such cross-linking might occur between microvilli o n different cells as well as bctween microvilli located on the same cell.
occupies the basolateral cell surfaces (unpublished observations). An intracellular location of C-CAM has been observed in granulocytes, megakaryocytes and In these cells, C-CAM is probably located to the membranes of intracellular granules. After platelet activation with AD P or collagen, which induces platelet aggregation, at least part of this C-CAM seems to be relocated to the cell surface(")).
Interaction with other components and regulation of C-CAM During the last decade, we have learned that cell adhesion is not a static but. rather, a highly dynamic
process that is regulated in various ways both during embryonic development and in several physiological and pathological Our knowledge about regulation of CAMs at the niolecular level and possible interactions of CAMs with other molecules in the cellular cortex is, however, still very limited. Yhosphorylation of C-CAM probably plays an important role in its regulation. C-CAM becomes phosphorylated on serine residues during culture of hepatocytcs(20),and a consensus sequence for CAMPdependent phosphorylation is present in thc cytoplasmic domain of C-CAM in rat liver(24’. Furthermore, Hubbard’s laboratory has demonstrated that a liver protein HA-4, which is identical to C-CAM, can be phosphorylated on tvrosine residues in an insulindependent manner(”). Another observation that C-CAM is under hormonal control was made in the uterine epithelia. Thus, the expression of C-CAM varies during the estrus cycle as a function o f the fluctuation of ovarian steroid hormones(”). Under the influence of progesterone, C-CAM becomes expressed in the glandular epithelium but is down-regulated in the luminal epithelium. Estrogen, on the other hand stimulates expression of C-CAM in the luminal epithelium. Other examples of regulation of C-CAM expression have been obtained from studies on liver development and regeneration. C-CAM appears rather late in the fetal development of the livei- and goes through transient down-regulation during regeneration after partial hepatectomy(”). In this context, it is also interesting to note that the expression of C-CAM is significantly altered in hepatocellular. carcinomas, so that it is either missing or chemically modified‘””.””. Another interesting obscrvation is that C-CAM can bind calmodulin in a specific, calcium-dependent manner(?’). This was discovered by Dr. Ingrid Blikstad (at the department of medical and physiological chemistry, University of Uppsala, Sweden), who has performed a detailed investigation of the binding interaction between C-CAM and calinodulin (Rlikstad, to be published). The calmodulin interaction may allow for a regulation of thc activity of C-CAM from the cytoplasmic side of the plasma membrane. Alternatively. adhesive interactions on the extracellular side niight influence the interaction with calmodulin on the cytoplasmic side. thereby regulating various intracellular processes. The interaction with calmodulin suggests that calcium ions may have a role in the cell biological function of C-CAM. An interesting question concerns the role of the putative ATPase activity of C-CAM. While the sequence published by Lin and Guidotti(24)should be that of liver ccto-ATPase, we have so far not been able to measure any ,4TPase activity in our purified preparations of C-CAM. However, the solubilization and removal of the protein from its natural environment may destroy its potential enzymatic activity. If C-CAM has ATPase activity, there is an interesting
possibility that this activity might influence its adhesive properties. In support of this idea, it has been reported that the adhesion of both chicken fibroblasts and mouse liver cells is inhibited by extracellular ATP(36,”). In our hands, ATP stimulated the aggregation of rat hepatocytcs. Furthermore. it has been demonstrated that hepatoina cells exhibit strong CAMP-regulated ectoATPase activity in cell-cell contact regions, but not on free cell surface areas(3‘). Thus, one might speculate of ATPt3’), that by controlled secretion C-CAM-carrying cells might be able to control their adheqive propertics viu interactions between ATP and C-CAM, and that the responsible ATPase activity might be regulated by phosphorylation . Finally, regulation of C-CAM‘s binding interactions could be brought about by site relocation, such as translocation from intraeellular to cell surface membranes upon platelet and granulocyte activation. Functional Aspects of C-CAM Both of the observations that there are at least two forms of C-CAM in liver and that the size of C-CAM varies amongst different tissues suggest that there are several isoforms of C-CAM. This is in agreement with the known diversity of molecules in the CEA gene family, but we do not know if the immunological dctection of C-CAM in the different tissues represent isoforms of C-CAM, or if some of these molccules are NCA or even CEA. In humans, all of these molecules cross-react immunologically@). The different subcellular locations of C-CAM in different cell types indicates that C-CAM does not mediate cell-cell binding under all conditions. However, although direct liomophilic binding has only been demonstrated for the liver form of C-CAM, available information so far indicates that a common denominator of C-CAM is participation in membrane-membrane binding that might be dynamically regulated. Thc picture that emerges is that C-CAM may participate in intercellular and cellular organizations at several levels. These include proper organization of hepatocytes in maturing liver, interactions between suprabasal cells in stratified epithelia, microvillar organization and dynamics in brush borders, plateletplatelet interactions in platelet aggregates, and interactions between vessel endothelial cells and leukocytes. It seems likely that the adhesive properties of the molccule play an important role in thcse situations. However, other functions of C-CAM cannot be excluded for the time being.
Functional Aspects of CAMs in General The research on cell adhesion has not only identified many different CAMs that are members of several protein families, but has also demonstrated that several distinct CAMs can be cxprcssed simultancously in one and the same cell type. Onc explanation for this apparent redundancy may be that it is part of a fail-safe
system that guarantees proper cellular organization even i l one of the system's components fails. It might also be possible that it is the combination of different CAMs in various ways that confers the tissue-specific adhesive properties on the respective cells. Another alternative is that different adhesion systems are parts of a network and that they cooperate in a more sophisticated way in the formation of various tissue structures. It is also likely that different CAMs have different functions. Furthermore, as mentioned in the Introduction. one should bear in mind that the CAM5 we know of today have been identified by tests that measure the ability to interfere with cell-cell binding in in v i m assays. This might mean that some CAMs could have other functions in addition to forming stablc cell-cell bond,. Some adhesion molecules, such as the desmogleins of desmosomes might primarily serve to create strong hue-stabilizing interccllular bonds. The cadherins also probably form strong intercellular bonds. in which their interactions with and organization of the contractile microfilaments play an important role. Other CAMs, however, may be principally involved in recognition and signalling between cells. Members of the Ig-superfamily may be particularly important in this respect. It is well-known that molecular interactions of the molecules of the Ig-superfamily in the immune system give rise to cellular si nals. which dramatically influence cellular behavioud' ). Maybe this also is true for CAMs of the Tg-superfamily outside the immune system.
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