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. 1983 Jan;80(2):430–434. doi: 10.1073/pnas.80.2.430

Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors

Coralie A Carothers Carraway *,†,‡, Goeh Jung †, Kermit L Carraway *,†
PMCID: PMC393391  PMID: 6572900

Abstract

The molecular nature of the cell surface-cytoskeleton interaction in microvilli isolated from ascites 13762 rat mammary adenocarcinoma sublines with immobile (MAT-C1) and mobile (MAT-B1) receptors was investigated by extraction and fractionation studies on the microvillar membranes. Extraction of membranes from MAT-C1 cells with Triton X-100-containing buffers gave insoluble residues showing three major components by NaDodSO4/polyacrylamide gel electrophoresis: actin, a 58,000-dalton polypeptide, and a cell surface glycoprotein of 75,000-80,000 daltons. The ratio of these components in Triton X-100-insoluble residues, as determined by scintillation counting of bands from gels of [3H]leucine-labeled microvillar membranes, approached equimolar, suggesting a specific complex of the components. The three components of the putative complex cosedimented on sucrose density gradients of Triton X-100/buffer-treated membranes. Gel filtration on Sepharose 2B gave a peak included in the column that contained only the glycoprotein, actin, and 58,000-dalton polypeptide by one-dimensional NaDodSO4 electrophoresis and by two-dimensional isoelectric focusing/NaDodSO4 electrophoresis. The glycoprotein-actin association could be disrupted only under strongly denaturing conditions. Complex prepared from MAT-B1 microvillar membranes by Sepharose 2B gel filtration in Triton X-100-containing buffers contained actin and the glycoprotein but no 58,000-dalton polypeptide. From these results we propose that the cell surface-cytoskeleton interactions in the 13762 tumor cell microvilli involve direct association of actin with the cell surface glycoprotein. We further suggest that the 58,000-dalton polypeptide stabilizes the association of this complex with the microfilaments in the MAT-C1 microvilli, thereby stabilizing the microvilli and restricting cell surface receptor mobility.

Keywords: isolated microvilli, microvillar membranes

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bourguignon L. Y., Singer S. J. Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5031–5035. doi: 10.1073/pnas.74.11.5031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branton D., Cohen C. M., Tyler J. Interaction of cytoskeletal proteins on the human erythrocyte membrane. Cell. 1981 Apr;24(1):24–32. doi: 10.1016/0092-8674(81)90497-9. [DOI] [PubMed] [Google Scholar]
  3. Carraway C. A., Cerra R. F., Bell P. B., Carraway K. L. Identification of a protein associated with both membrane and cytoskeleton fractions from branched but not unbranched microvilli of 13762 rat mammary adenocarcinoma ascites tumor sublines. Biochim Biophys Acta. 1982 Oct 28;719(1):126–139. doi: 10.1016/0304-4165(82)90316-6. [DOI] [PubMed] [Google Scholar]
  4. Carraway K. L., Cerra R. F., Jung G., Carraway C. A. Membrane-associated actin from the microvillar membranes of ascites tumor cells. J Cell Biol. 1982 Sep;94(3):624–630. doi: 10.1083/jcb.94.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carraway K. L., Doss R. C., Huggins J. W., Chesnut R. W., Carraway C. A. Effects of cytoskeletal perturbant drugs on ecto 5'-nucleotidase, a concanavalin A receptor. J Cell Biol. 1979 Dec;83(3):529–543. doi: 10.1083/jcb.83.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carraway K. L., Huggins J. W., Cerra R. F., Yeltman D. R., Carraway C. A. alpha-Actinin-containing branched microvilli isolated from an ascites adenocarcinoma. Nature. 1980 Jun 12;285(5765):508–510. doi: 10.1038/285508a0. [DOI] [PubMed] [Google Scholar]
  7. Heidmann T., Changeux J. P. Structural and functional properties of the acetylcholine receptor protein in its purified and membrane-bound states. Annu Rev Biochem. 1978;47:317–357. doi: 10.1146/annurev.bi.47.070178.001533. [DOI] [PubMed] [Google Scholar]
  8. Huggins J. W., Trenbeath T. P., Yeltman D. R., Carraway K. L. Restricted concanavalin A redistribution on the branched microvilli of an ascites tumor subline. Exp Cell Res. 1980 May;127(1):31–46. doi: 10.1016/0014-4827(80)90412-7. [DOI] [PubMed] [Google Scholar]
  9. King J., Laemmli U. K. Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol. 1971 Dec 28;62(3):465–477. doi: 10.1016/0022-2836(71)90148-3. [DOI] [PubMed] [Google Scholar]
  10. Koch G. L., Smith M. J. An association between actin and the major histocompatibility antigen H-2. Nature. 1978 May 25;273(5660):274–278. doi: 10.1038/273274a0. [DOI] [PubMed] [Google Scholar]
  11. Mescher M. F., Jose M. J., Balk S. P. Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature. 1981 Jan 15;289(5794):139–144. doi: 10.1038/289139a0. [DOI] [PubMed] [Google Scholar]
  12. Nicolson G. L. Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over surface components. Biochim Biophys Acta. 1976 Apr 13;457(1):57–108. doi: 10.1016/0304-4157(76)90014-9. [DOI] [PubMed] [Google Scholar]
  13. Painter R. G., Ginsberg M. Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton. J Cell Biol. 1982 Feb;92(2):565–573. doi: 10.1083/jcb.92.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pober J. S., Guild B. C., Strominger J. L., Veatch W. R. Purification of HLA-A2 antigen, fluorescent labeling of its intracellular region, and demonstration of an interaction between fluorescently labeled HLA-A2 antigen and lymphoblastoid cell cytoskeleton proteins in vitro. Biochemistry. 1981 Sep 15;20(19):5625–5633. doi: 10.1021/bi00522a042. [DOI] [PubMed] [Google Scholar]
  15. Rubin R. W., Milikowski C. Over two hundred polypeptides resolved from the human erythrocyte membrane. Biochim Biophys Acta. 1978 May 4;509(1):100–110. doi: 10.1016/0005-2736(78)90011-1. [DOI] [PubMed] [Google Scholar]
  16. Sheetz M. P. DNase-I-dependent dissociation of erythrocyte cytoskeletons. J Cell Biol. 1979 Apr;81(1):266–270. doi: 10.1083/jcb.81.1.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sherblom A. P., Carraway K. L. A complex of two cell surface glycoproteins from ascites mammary adenocarcinoma cells. J Biol Chem. 1980 Dec 25;255(24):12051–12059. [PubMed] [Google Scholar]
  18. Sheterline P., Hopkins C. R. Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes. J Cell Biol. 1981 Sep;90(3):743–754. doi: 10.1083/jcb.90.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Strauch A. R., Luna E. J., LaFountain J. R., Jr Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm. J Cell Biol. 1980 Jul;86(1):315–325. doi: 10.1083/jcb.86.1.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. de Petris S. Preferential distribution of surface immunoglobulins on microvilli. Nature. 1978 Mar 2;272(5648):66–68. doi: 10.1038/272066a0. [DOI] [PubMed] [Google Scholar]

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