Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice.

Integrins are heterodimeric transmembrane glycoproteins which are engaged in a variety of cellular functions, such as adhesion, migration and differentiation1. The integrin alpha 6 beta 4 is expressed on squamous epithelia, on subsets of endothelial cells, immature thymocytes and on Schwann cells and fibroblasts in the peripheral nervous system. In stratified epithelia, alpha 6 beta 4 is concentrated in specialised adhesion structures, called hemidesmosomes, which are implicated in the stable attachment of the basal cells to the underlying basement membrane by connecting the intermediate filaments with the extracellular matrix. The nature of the interactions between the various hemidesmosomal proteins, that lead to the formation of hemidesmosome is poorly understood. To study the contribution of the integrin alpha 6 beta 4 in hemidesmosome formation and their anchoring properties, we inactivated the beta 4 gene in mice by targeted gene disruption. Homozygous beta 4 null mice died shortly after birth and displayed extensive detachment of the epidermis and other squamous epithelia. The dramatically reduced adhesive properties of the skin was accompanied by the absence of hemidesmosomes at the basal surface of keratinocytes. No evidence was found for impaired T-cell development, nor for defects in myelination in the peripheral nervous system.

Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids.

The precursor protein of von Willebrand factor (pro-vWF) consists of four different repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, followed by a carboxy-terminal region of 151 amino acids without obvious internal homology. Previously, we have shown the requirement of the domains D1, D2, D', and D3 of pro-vWF in the assembly of pro-vWF dimers into multimers. Here, we define the domains of vWF involved in dimerization, using deletion mutants of full-length vWF cDNA transiently expressed in monkey kidney COS-1 cells. It is shown that only the carboxy-terminal 151 amino acid residues of vWF are required for dimerization. In addition, by analyzing a construct, encoding only the carboxy-terminal 151 amino acids of vWF, we find that the formation of dimers is an event independent of other domains present on pro-vWF, such as the domains C1 and C2 previously suggested to be involved in dimerization. Furthermore, it is shown that a deletion mutant of vWF, lacking the carboxy-terminal 151 amino acid residues and thus unable to dimerize, is proteolytically degraded in the ER. In contrast, a mutant protein, composed only of the carboxy-terminal 151 amino acids of vWF, and able to dimerize, is transported from the ER in a similar fashion as wild-type vWF. The role of the ER in the assembly of vWF is discussed with regard to the data presented in this paper on the intracellular fate of several vWF mutant proteins.

Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development.

Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described. While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A. We show that alpha6A-/- mice develop normally and are fertile. The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in alpha6A-/- mice. However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.

Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface.

Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes.

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.

Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity.

Multivesicular bodies are endocytic compartments containing multiple small vesicles that originate from the invagination and 'pinching off' of the limiting membrane into the luminal space [1] [2] [3]. The molecular mechanisms responsible for the formation of these compartments are unknown. In the human melanoma cell line Mel JuSo, newly synthesised major histocompatibility complex (MHC) class II molecules accumulate in multivesicular early lysosomes [4]. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin induced the transient vacuolation of early MHC class II compartments, but also of early and late endosomes. We demonstrate that endocytic membrane influx is required for the wortmannin-induced swelling of vesicles. The wortmannin-induced vacuoles contained a reduced number of intraluminal vesicles that were linked to the limiting membrane by membraneous connections. These data suggest that wortmannin inhibits the invagination and/or pinching off of intraluminal vesicles and provide evidence of a role for PI 3-kinase in multivesicular body morphogenesis. We propose that the wortmannin-induced vacuolation occurs as a result of the inability of multivesicular bodies to store endocytosed membranes as intraluminal vesicles thereby causing the formation of large 'empty' vacuoles.

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.

Gap junction protein connexin-43 interacts directly with microtubules.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.

Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.

The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting.

Activation of Rac1, a member of the Rho family of GTPases, is associated with multiple cellular responses, including membrane ruffling and focal complex formation. The mechanisms by which Rac1 is coupled to these functional responses are not well understood. It was recently shown that ARF6, a GTPase implicated in cytoskeletal alterations and a membrane recycling pathway, is required for Rac1-dependent phagocytosis in macrophages (Q. Zhang et al., J. Biol. Chem. 273:19977-19981, 1998). To determine whether ARF6 is required for Rac1-dependent cytoskeletal responses in macrophages, we expressed wild-type (WT) or guanine nucleotide binding-deficient alleles (T27N) of ARF6 in macrophages coexpressing activated alleles of Rac1 (Q61L) or Cdc42 (Q61L) or stimulated with colony-stimulating factor 1 (CSF-1). Expression of ARF6 T27N but not ARF6 WT inhibited ruffles mediated by Rac1 Q61L or CSF-1. In contrast, expression of ARF6 T27N did not inhibit Rac1 Q61L-mediated focal complex formation and did not impair Cdc42 Q61L-mediated filopodial formation. Cryoimmunogold electron microscopy demonstrated the presence of ARF6 in membrane ruffles induced by either CSF-1 or Rac1 Q61L. Addition of CSF-1 to macrophages led to the redistribution of ARF6 from the interior of the cell to the plasma membrane, suggesting that this growth factor triggers ARF6 activation. Direct targeting of Rac1 to the plasma membrane did not bypass the blockade in ruffling induced by ARF6 T27N, indicating that ARF6 regulates a pathway leading to membrane ruffling that occurs after the activation and membrane association of Rac. These data demonstrate that intact ARF6 function is required for coupling activated Rac to one of several effector pathways and suggest that a principal function of ARF6 is to coordinate Rac activation with plasma membrane-based protrusive events.

Antibody-induced modulation and shedding of mammary tumor virus antigens on the surfaces of GR ascites leukemia cells as compared with normal antigens.

The distribution, antibody-induced redistribution, and shedding of murine mammary tumor virus (MuMTV) antigens and the surfaces of GR mouse ascites leukemia (GRSL) cells were studied by the immunoferritin technique and compared with the same activities of thy 1.2 and H-2.8 antigens. MuMTV antigens were redistributed easily and then largely shed from the cell surface; in contrast, H-2.8 antigen moved easily and probably was partially released from the plasms membrane and Thy 1.2 antigen moved slowly and was some what interiorized. The complement-dependent cytotoxicity test was used to study the possibility of antigenic modulation for these cell-surface antigens from the surface of the GRSL cells could be modulated by preincubation with anti-MuMTV serum, in contrast to H-2.8 and Thy 1.2 antigens. The results obtained with the immunoferritin technique and the cytotoxicity test correlated well and sug-ested that the shedding of MuMTV antigens from the cell surfaces may occur in vivo, providing the tumor a way to escape from the immune defense of the host. Thy 1.2 and H-2.8 antigens were present on the envolope of B and C particles, which suggested that these viruses do not select a Thy 1.2 or H-2.8-negative area of the GRSL cell surface as amaturation site.

Immunologic, virologic, and genetic aspects of mammary tumor virus-induced cell-surface antigens: presence of these antigens and the Thy 1.2 antigen on murine mammary gland and tumor cells.

The distribution of the normal differentiation antigen Thy 1 and the mammary tumor virus (MTV)-induced antigens or antigen complexes MLm and MLr were studied in mouse mammary gland cells, mammary tumor cells, and other cell types, by use of ascites leukemia cells of the GR mouse strain as target cells in the cytotoxicity test. The Thy 1.2 antigen was detected by an AKR antiserum to C3Hf thymocytes. MLm was shown by a homologous C57BL antiserum to GRSL2 leukemia (absorbed in vivo in GR mice); MLr was detected by a rabbit heterologous antiserum (absorbed in vivo in C57BL or GR mice and in vitro with BALB/c milk) prepared against Tween 80- and ether-treated purified B particles. Sera from Sprague-Dawley rats bearing murine leukemia virus (MuLV)-producing syngeneic tumors were not cytotoxic or only slightly cytotoxic for GR leukemias transplanted in vivo, which indicated that MuLV-induced antigens were absent or present in very low quantity in such leukemias. The MLr and MLn antigens or antigen complexes were possibly identical to the mammary leukemia (ML) antigen, since they could be detected not only on GR but also on DBA/2 leukemia cells and since their distribution was exactly the same as that of MTV. Both the MLr and MLm antigens were present in purified B particles, and antigenic activity were present in purified B particles, and antigenic activity was enhanced by destruction of the purified virus particles. The antigens were about eightfold enriched in a preparation of B-particle envelopes, as shown by quantitative cytotoxicity absorption (CYTA) tests. Purified nucleoid fractions of B particles were only lightly positive for the antigen, probably due to envelope contamination. One dominant gene was responsible for the expression of MLr, as shown by CYTA tests with mammary glands of individual animals of segregating crosses between the GR strain with high mammary cancer incidence and strains with low incidence. This gene was closely linked with or was possibly identical to 1) the gene for cytoplasmic MTV gs antigen expression as seen by fixed cell immunofluorescence, and 2) the gene causing mammary tumors in the GR mouse strain.

In vitro differentiation and progression of mouse mammary tumor cells.

We have isolated clonal cell lines from a transplanted adenocarcinoma induced by the RIII strain of mouse mammary tumor virus in a BALB/c mouse. Three major morphological cell types of these lines are developmentally linked; polygonal cells give rise to cuboidal and then to elongated cells. All cell lines expressed markers that are characteristic of mammary basal cells. In addition, the polygonal lines contained cells that have cell markers and ultrastructural features of epithelial cells; in these lines an occasional cell was found with myoepithelial features. The cuboidal and elongated lines lacked many epithelial differentiation characteristics and showed no myoepithelial differentiation. The cell lines contained variable numbers of acquired mouse mammary tumor virus and ecotropic murine leukemia virus proviruses. The various subclones derived from the original cell lines contained, in addition to the acquired proviruses of the parental line, one or more unique proviruses of either mouse mammary tumor virus or ecotropic murine leukemia virus origin. These unique insertions were used as genotypic markers to demonstrate the clonal relationship of the cell lines. Both polygonal and elongated cells are tumorigenic and give rise to adenocarcinomas and sarcoma-like tumors, respectively. In contrast, the cuboidal cells are poorly tumorigenic. Since cuboidal cells are derived from the polygonal cells, this suggests that tumor progression in this system proceeds via intermediates that are either poorly or nontumorigenic.

Endocytosis and intracellular routing of an antibody-ricin A chain conjugate.

An immunotoxin (IT) was prepared from monoclonal antibody (MoAb) 115D8 and ricin A chain. MoAb 115D8 is directed against the carcinoma-associated sialomucin MAM-6. In a protein synthesis inhibition assay this IT was cytotoxic for the human breast cancer cell line T47D. Using postembedding immunoelectron microscopy the binding and intracellular routing of the IT in T47D cells were studied by simultaneous labeling of both IT moieties, MoAb and A chain, and compared with the fate of native ricin and MoAb 115D8. The IT was internalized into the cell by two different pathways: one via coated pits-coated vesicles followed by transport to the lysosomes and one via large enclosed invaginations of the plasma membrane which apparently fused with lysosomes. This internalization was similar to the endocytosis of MoAb 115D8. During transport via both pathways the IT remained intact until it reached the lysosomes as suggested by the observation that the labels for 115D8 and ricin A chain remained closely associated. Moreover, in areas with abundant endocytic vesicles the labels for both IT moieties were also found in the cytosol, suggesting that intact IT is translocated from the vesicles into the cytosol. In control experiments, native ricin, but not unconjugated MoAb 115D8, was found in the cytosol after internalization. Data presented here show for the first time the complete intracellular pathway of an antibody-ricin A chain conjugate, including the translocation of the A toxin subunit into the cytosol. This IT may be useful for therapy of those tumors which express a high level of MAM-6 on the cell surface.

Levels of mammary tumor virus in hormone-dependent and -independent mouse mammary tumor cells.

Levels of mammary tumor virus particles (types A and B) and levels of the virus antigen were assayed in hormone-dependent and -independent mammary tumors of GR mice. Various transplant generations of seven separate tumor lines were investigated. The results indicated that the tumors consisted of different cell clones, each of which exhibited a separate progressive expression and subsequent loss of the mammary tumor virus. When the tumors were transplanted, levels of B particles first declined in the hormone-dependent cells, but in later transplant generations, the B particle content of the autonomous cells also dropped. In some tumor lines, this was accompanied by a decrease in viral antigens and/or A particles, but in other lines these concentrations remained high. One tumor line (line V) that remained hormone-dependent throughout nine transplantations was practically devoid of B particles but contained high levels of A particles and mammary tumor antigen.

Hepatocyte surface molecule involved in the adhesion of TA3 mammary carcinoma cells to rat hepatocyte cultures.

To identify adhesion molecules involved in the formation of liver metastases, we prepared monoclonal antibodies against rat liver plasma membranes, that inhibited the adhesion of mouse metastatic TA3 mammary carcinoma cells to rat hepatocytes in vitro. Two such antibodies (designated OPAR-1 and OPAR-2) were obtained, that inhibited by over 70%. As assessed with gel electrophoresis and immunoblotting, these antibodies bound predominantly to plasma membrane proteins with molecular weights of 125,000 and 100,000. Using immunoelectron microscopy, the OPAR antigen was found to be abundantly present on the sinusoidal surface of hepatocytes, and in addition on contiguous hepatocyte surfaces and Kupffer cells, but was absent from sinusoidal endothelial cells. The tissue distribution and molecular weight indicate that the OPAR antigen is different from other hepatic adhesion molecules. Adhesion of MB6A lymphosarcoma cells was not inhibited by OPAR antibodies, indicating that these cells adhere to hepatocytes via a different surface molecule.

Structural heterogeneity of a human melanoma-associated antigen.

The biosynthesis, structure, and topology of a melanoma-associated antigen, previously defined with the monoclonal antibody NKI/C-3 was studied. A polyclonal rabbit antiserum was raised against the antigen with a broader reactivity than the previously used monoclonal antibody NKI/C-3. The antigen was shown to consist of a single protein backbone to which two or three N-linked glycans were added cotranslationally. Extensive further heterogeneity was generated in the Golgi compartment and was shown to be dependent on the presence of complex type sugars. Although the antigen is associated with melanomas, it was not codistributed with the tyrosinase activity associated with melanogenesis. The antigen did show codistribution with cathepsin D, which is a marker for lysosomal functions.