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. 1986 Oct 1;103(4):1441–1450. doi: 10.1083/jcb.103.4.1441

A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies

PMCID: PMC2114326  PMID: 3533954

Abstract

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane- bound plaques of the fascia adherens. In cultured chick lens cells, A- CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A- binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451- 1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction- specific membrane glycoprotein that is involved in intercellular adhesion in these sites.

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Selected References

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  1. Alcalá J., Maisel H., Lieska N. The presence of delta-crystallin in the plasma membrane of chick lens fiber cells. Exp Cell Res. 1977 Oct 1;109(1):63–69. doi: 10.1016/0014-4827(77)90044-1. [DOI] [PubMed] [Google Scholar]
  2. Avnur Z., Geiger B. The removal of extracellular fibronectin from areas of cell-substrate contact. Cell. 1981 Jul;25(1):121–132. doi: 10.1016/0092-8674(81)90236-1. [DOI] [PubMed] [Google Scholar]
  3. Behrens J., Birchmeier W., Goodman S. L., Imhof B. A. Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin. J Cell Biol. 1985 Oct;101(4):1307–1315. doi: 10.1083/jcb.101.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boller K., Vestweber D., Kemler R. Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells. J Cell Biol. 1985 Jan;100(1):327–332. doi: 10.1083/jcb.100.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  6. Burridge K. Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Methods Enzymol. 1978;50:54–64. doi: 10.1016/0076-6879(78)50007-4. [DOI] [PubMed] [Google Scholar]
  7. Cohen S. M., Gorbsky G., Steinberg M. S. Immunochemical characterization of related families of glycoproteins in desmosomes. J Biol Chem. 1983 Feb 25;258(4):2621–2627. [PubMed] [Google Scholar]
  8. Cunningham B. A., Leutzinger Y., Gallin W. J., Sorkin B. C., Edelman G. M. Linear organization of the liver cell adhesion molecule L-CAM. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5787–5791. doi: 10.1073/pnas.81.18.5787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Damsky C. H., Richa J., Solter D., Knudsen K., Buck C. A. Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue. Cell. 1983 Sep;34(2):455–466. doi: 10.1016/0092-8674(83)90379-3. [DOI] [PubMed] [Google Scholar]
  10. Duguid J. R., Revel J. P. The protein components of the gap junction. Cold Spring Harb Symp Quant Biol. 1976;40:45–47. doi: 10.1101/sqb.1976.040.01.007. [DOI] [PubMed] [Google Scholar]
  11. Edelman G. M. Cell adhesion molecules. Science. 1983 Feb 4;219(4584):450–457. doi: 10.1126/science.6823544. [DOI] [PubMed] [Google Scholar]
  12. FARQUHAR M. G., PALADE G. E. Junctional complexes in various epithelia. J Cell Biol. 1963 May;17:375–412. doi: 10.1083/jcb.17.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fawcett D. W., McNutt N. S. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969 Jul;42(1):1–45. doi: 10.1083/jcb.42.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Folkman J., Moscona A. Role of cell shape in growth control. Nature. 1978 Jun 1;273(5661):345–349. doi: 10.1038/273345a0. [DOI] [PubMed] [Google Scholar]
  15. Gallin W. J., Edelman G. M., Cunningham B. A. Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1038–1042. doi: 10.1073/pnas.80.4.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  17. Geiger B., Dutton A. H., Tokuyasu K. T., Singer S. J. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol. 1981 Dec;91(3 Pt 1):614–628. doi: 10.1083/jcb.91.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geiger B. Involvement of vinculin in contact-induced cytoskeletal interactions. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):671–682. doi: 10.1101/sqb.1982.046.01.063. [DOI] [PubMed] [Google Scholar]
  19. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  20. Geiger B., Schmid E., Franke W. W. Spatial distribution of proteins specific for desmosomes and adhaerens junctions in epithelial cells demonstrated by double immunofluorescence microscopy. Differentiation. 1983;23(3):189–205. doi: 10.1111/j.1432-0436.1982.tb01283.x. [DOI] [PubMed] [Google Scholar]
  21. Geiger B., Singer S. J. Association of microtubules and intermediate filaments in chicken gizzard cells as detected by double immunofluorescence. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4769–4773. doi: 10.1073/pnas.77.8.4769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Geiger B., Singer S. J. The participation of alpha-actinin in the capping of cell membrane components. Cell. 1979 Jan;16(1):213–222. doi: 10.1016/0092-8674(79)90202-2. [DOI] [PubMed] [Google Scholar]
  23. Geiger B., Volk T., Volberg T. Molecular heterogeneity of adherens junctions. J Cell Biol. 1985 Oct;101(4):1523–1531. doi: 10.1083/jcb.101.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goodenough D. A. Bulk isolation of mouse hepatocyte gap junctions. Characterization of the principal protein, connexin. J Cell Biol. 1974 May;61(2):557–563. doi: 10.1083/jcb.61.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gorbsky G., Steinberg M. S. Isolation of the intercellular glycoproteins of desmosomes. J Cell Biol. 1981 Jul;90(1):243–248. doi: 10.1083/jcb.90.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Grinnell F. Cellular adhesiveness and extracellular substrata. Int Rev Cytol. 1978;53:65–144. doi: 10.1016/s0074-7696(08)62241-x. [DOI] [PubMed] [Google Scholar]
  27. Hatta K., Okada T. S., Takeichi M. A monoclonal antibody disrupting calcium-dependent cell-cell adhesion of brain tissues: possible role of its target antigen in animal pattern formation. Proc Natl Acad Sci U S A. 1985 May;82(9):2789–2793. doi: 10.1073/pnas.82.9.2789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Heggeness M. H., Ash J. F., Singer S. J. Transmembrane linkage of fibronectin to intracellular actin-containing filaments in cultured human fibroblasts. Ann N Y Acad Sci. 1978 Jun 20;312:414–417. doi: 10.1111/j.1749-6632.1978.tb16822.x. [DOI] [PubMed] [Google Scholar]
  29. Hertzberg E. L., Gilula N. B. Isolation and characterization of gap junctions from rat liver. J Biol Chem. 1979 Mar 25;254(6):2138–2147. [PubMed] [Google Scholar]
  30. Hyafil F., Morello D., Babinet C., Jacob F. A cell surface glycoprotein involved in the compaction of embryonal carcinoma cells and cleavage stage embryos. Cell. 1980 Oct;21(3):927–934. doi: 10.1016/0092-8674(80)90456-0. [DOI] [PubMed] [Google Scholar]
  31. Hynes R. O., Destree A. T., Wagner D. D. Relationships between microfilaments, cell-substratum adhesion, and fibronectin. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):659–670. doi: 10.1101/sqb.1982.046.01.062. [DOI] [PubMed] [Google Scholar]
  32. Kartenbeck J., Schmid E., Franke W. W., Geiger B. Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material. EMBO J. 1982;1(6):725–732. doi: 10.1002/j.1460-2075.1982.tb01237.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Maher P. A., Singer S. J. Anomalous interaction of the acetylcholine receptor protein with the nonionic detergent Triton X-114. Proc Natl Acad Sci U S A. 1985 Feb;82(4):958–962. doi: 10.1073/pnas.82.4.958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  36. Mueller H., Franke W. W. Biochemical and immunological characterization of desmoplakins I and II, the major polypeptides of the desmosomal plaque. J Mol Biol. 1983 Feb 5;163(4):647–671. doi: 10.1016/0022-2836(83)90116-x. [DOI] [PubMed] [Google Scholar]
  37. Ocklind C., Obrink B. Intercellular adhesion of rat hepatocytes. Identification of a cell surface glycoprotein involved in the initial adhesion process. J Biol Chem. 1982 Jun 25;257(12):6788–6795. [PubMed] [Google Scholar]
  38. Peyriéras N., Hyafil F., Louvard D., Ploegh H. L., Jacob F. Uvomorulin: a nonintegral membrane protein of early mouse embryo. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6274–6277. doi: 10.1073/pnas.80.20.6274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shirayoshi Y., Okada T. S., Takeichi M. The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. Cell. 1983 Dec;35(3 Pt 2):631–638. doi: 10.1016/0092-8674(83)90095-8. [DOI] [PubMed] [Google Scholar]
  40. Singer I. I. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell. 1979 Mar;16(3):675–685. doi: 10.1016/0092-8674(79)90040-0. [DOI] [PubMed] [Google Scholar]
  41. Staehelin L. A. Structure and function of intercellular junctions. Int Rev Cytol. 1974;39:191–283. doi: 10.1016/s0074-7696(08)60940-7. [DOI] [PubMed] [Google Scholar]
  42. Thiery J. P., Duband J. L., Rutishauser U., Edelman G. M. Cell adhesion molecules in early chicken embryogenesis. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6737–6741. doi: 10.1073/pnas.79.21.6737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tokuyasu K. T., Dutton A. H., Geiger B., Singer S. J. Ultrastructure of chicken cardiac muscle as studied by double immunolabeling in electron microscopy. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7619–7623. doi: 10.1073/pnas.78.12.7619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  45. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vasiliev J. M., Gelfand I. M. Mechanisms of morphogenesis in cell cultures. Int Rev Cytol. 1977;50:159–274. doi: 10.1016/s0074-7696(08)60099-6. [DOI] [PubMed] [Google Scholar]
  47. Volberg T., Geiger B., Kartenbeck J., Franke W. W. Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions. J Cell Biol. 1986 May;102(5):1832–1842. doi: 10.1083/jcb.102.5.1832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Volk T., Geiger B. A 135-kd membrane protein of intercellular adherens junctions. EMBO J. 1984 Oct;3(10):2249–2260. doi: 10.1002/j.1460-2075.1984.tb02123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Volk T., Geiger B. A-CAM: a 135-kD receptor of intercellular adherens junctions. II. Antibody-mediated modulation of junction formation. J Cell Biol. 1986 Oct;103(4):1451–1464. doi: 10.1083/jcb.103.4.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yoshida-Noro C., Suzuki N., Takeichi M. Molecular nature of the calcium-dependent cell-cell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody. Dev Biol. 1984 Jan;101(1):19–27. doi: 10.1016/0012-1606(84)90112-x. [DOI] [PubMed] [Google Scholar]

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